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Avenanthramide-C Restores Impaired Plasticity and Cognition in Alzheimer’s

Disease Model Mice

Article  in  Molecular Neurobiology · January 2020

DOI: 10.1007/s12035-019-01707-5

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Molecular Neurobiology
https://doi.org/10.1007/s12035-019-01707-5

Avenanthramide-C Restores Impaired Plasticity and Cognition

in Alzheimer’s Disease Model Mice
Vijay Sankar Ramasamy 1,2 & Manikandan Samidurai 1,2 & Hyung Joon Park 3 & Ming Wang 1,2 & Ra Young Park 4 &
Seon Young Yu 5 & Hee Kyung Kang 1 & Semi Hong 3 & Won-Seok Choi 3 & Yu Young Lee 6 & Hyung-Seok Kim 7,8 &
Jihoon Jo 1,2,9,10

Received: 23 April 2019 / Accepted: 10 July 2019

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by cognitive decline and dementia with no
effective treatment. Here, we investigated a novel compound from oats named avenanthramide-C (Avn-C), on AD-related
memory impairment and behavioral deficits in transgenic mouse models. Acute hippocampal slices of wild-type or AD trans-
genic mice were treated with Avn-C in the presence or absence of oligomeric Aβ42. LTP analyses and immunoblotting were
performed to assess the effect of Avn-C on Aβ-induced memory impairment. To further investigate the effect of Avn-C on
impaired memory and Aβ pathology, two different AD transgenic mice (Tg2576 and 5XFAD) models were orally treated with
either Avn-C or vehicle for 2 weeks. They were then assessed for the effect of the treatment on neuropathologies and behavioral
impairments. Avn-C reversed impaired LTP in both ex vivo- and in vivo-treated AD mice hippocampus. Oral administration
(6 mg/kg per day) for 2 weeks in AD mice leads to improved recognition and spatial memory, reduced caspase-3 cleavage,
reversed neuroinflammation, and to accelerated glycogen synthase kinase-3β (pS9GSK-3β) and interleukin (IL-10) levels. Avn-
C exerts its beneficial effects by binding to α1A adrenergic receptors to stimulate adenosine monophosphate-activated kinase
(AMPK). All of the beneficial effects of Avn-C on LTP retrieval could be blocked by prazosin hydrochloride, a specific inhibitor
of α1A adrenergic receptors. Our findings provide evidence, for the first time, that oats’ Avn-C reverses the AD-related memory

Vijay Sankar Ramasamy, Manikandan Samidurai and Hyung Joon Park

contributed equally to this work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12035-019-01707-5) contains supplementary
material, which is available to authorized users.

* Won-Seok Choi
* Yu Young Lee
* Hyung-Seok Kim
* Jihoon Jo
Jihoon.Jo@chonnam.ac.kr
1
NeuroMedical Convergence Laboratory, Biomedical Research
Institute, Chonnam National University Hospital, Jebong-ro,
Gwangju 501-757, Republic of Korea
2
Department of Biomedical Sciences, BK21 PLUS Center for
Creative Biomedical Scientists at Chonnam National University,
Research Institute of Medical Sciences, Chonnam National
University Medical School, Gwangju 501-757, Republic of Korea
3 10
School of Biological Sciences and Technology, College of Natural
Sciences, Chonnam National University, Gwangju 500-757,
Republic of Korea
4
Bio-Medical treatment technology development projects, Institute for
Biomedical Science, Chonnam National University Hwasun
Hospital, Gwangju, Republic of Korea
5
Forensic Medicine Division, National Forensic Service Daegu
Institute, Daegu 39872, Republic of Korea
6
Department of Central Area, National Institute of Crop Science,
Rural Development Administration, Suwon 16429, Republic of
Korea
7
Department of Forensic Medicine, Chonnam National University
Medical School, Gwangju, South Korea
8
Center for Creative Biomedical Scientists, Chonnam National
University Medical School, Gwangju 501-757, Republic of Korea
9
Department of Neurology, Chonnam National University Medical
School, Gwangju 501-757, Republic of Korea
NeuroMedical Convergence Laboratory, Department of Biomedical
Sciences and Neurology, Chonnam National University Medical
School, Gwangju 501-746, Republic of Korea

and behavioral impairments, and establish it as a potential candidate for Alzheimer’s disease drug development.
Keywords Alzheimer’s disease . Avn-C . Oats . LTP . Amyloid-β . Memory . Mouse model
Introduction
Alzheimer’s disease (AD) is the most common form of de-
mentia in the elderly population worldwide, characterized by
the memory and cognition impairment. Though there are sev-
eral mechanisms and factors proposed for AD condition, β-
amyloid is thought to be the major risk factor, driving oxida-
tive stress, tau hyperphosphorylation, inflammation, apoptotic
signaling, and neuronal loss [1, 2]. Acetylcholinesterase inhib-
itors and an N-methyl-d-aspartate receptor blocker represent
the few currently available drugs approved for AD treatment,
but these drugs are minimally efficacious and have adverse
effects, limiting their use [3, 4]. Many clinical trials targeting dioxide-induced neuronal degeneration and serum TNFα levels
Aβ with monoclonal antibodies or small peptide inhibitors
have failed because their effects were too limited [5].
A critical early event in AD is the synaptic dysfunction and
impaired synaptic plasticity. Spatial learning and memory im-
pairment, an early clinical feature of AD, is caused by synaptic
dysfunction rather than neuronal loss in AD transgenic mice
[6]. Soluble oligomeric form of Aβ activates a variety of mo-
lecular cascades that culminate in early synapse dysfunction.
Acute application of oligomeric Aβ42 to hippocampal slices
inhibits long-term potentiation (LTP)—a form of synaptic
plasticity, by activating caspase-3 and GSK3β [7], reducing
NMDA receptor surface expression [8] and increasing AMPA
receptors internalization [9]. In addition, Aβ induce synaptic
dysfunction by promoting pro-inflammatory cytokine produc-
tion. Mouse hippocampal slices exposed to Aβ oligomers
shown to increase the production of tumor necrosis factor-α
(TNFα) and reduction in LTP. In support of this, increased
levels of pro-inflammatory cytokines such as interleukin-1β
(IL1β) [10], interleukin-6 (IL-6) [11], and TNFα [12] have
been observed in brain samples from both AD patients and
AD mice models. Aberrant microglial activation and cytokine
production have been correlated with cognitive decline [13,
14]. Furthermore, in an AD mouse model of tauopathy,
microglial activation coincided with hippocampal synapse
loss and impaired synaptic function prior to the onset of fibril-
lary tau tangles [15, 16]. Such observations highlight targeting
these pathways would be beneficial in AD treatment. In sup-
port of this, administration of nonsteroidal anti-inflammatory
drugs ameliorates Aβ pathogenesis and memory impairment
[17], although efficacy in clinical trials remains to be shown.
In searches for new anti-AD drugs, natural products have
become an important avenue because of their unique composi-
tion of therapeutic compounds, for example epigallocatechin
gallate (green tea component), gallic acid, resveratrol, and
curcumin were identified as multifunctional compounds with
anti-AD properties, yet their poor bioavailability limits their
therapeutic effects [18–21]. Here, we report the effects of
Mol Neurobiol
avenanthramide-C (Avn-C), a low molecular weight polyphe-
nolic compound exclusively found in oats (Avena sativa), on
synaptic dysfunction and memory impairment in Tg2576 and
5XFAD mouse models of AD. Previous studies have shown
that Avn-C have the anti-inflammatory effects of decreasing
pro-inflammatory markers (IL-6 and TNFα) through decreased
NF-κB activity [22–24]. Rats fed with Avn-C supplemented
diet for 50 days display lower reactive oxygen species genera-
tion and lipid peroxidation in skeletal muscle [25]. Also,
8 weeks of Avn-C supplementation in women abolishes down-
hill running-induced inflammation [26]. Furthermore, orally
administered avenanthramides are shown to reduce titanium
in Sprague-Dawley rats [27]. Based on these observations, we
hypothesized that the cytoprotective properties of Avn-C would
be beneficial for AD-related memory impairment.
Materials and Methods
Mouse Models
Wild-type (C57BL/6J, 7–8 months old), Tg2576 (APP
KM670/671NL, 7–8 months old, Taconic), and 5XFAD
(APP KM670/671NL [Swedish], APP I716V [Florida], APP
V717I [London], PSEN1 M146L, PSEN1 L286V, 5–6 months
old, Jackson Laboratory) mice were used for the experiments.
In all experiments, male mice were used. Animals were housed
in individually ventilated cages with free access to food and
water. The breeding room was controlled with a 12-h light/dark
cycle (lights on at 8:00 p.m.) and the temperature maintained at
22–30 °C. All experiments involving animals followed ap-
proved protocols from the Institutional Animal Care and Use
Committee of Chonnam National University Medical School,
Republic of Korea. In the study, n refers to the number of
animals used in any particular experiment or the number of
individual replicates (in vitro).
Hippocampal Slice Preparation and Avn-C Treatment
Animals were sacrificed between 9:00 and 10:00 a.m. by cer-
vical dislocation. After cervical dislocation, the brain was
quickly removed and transferred to ice-cold artificial cerebro-
spinal fluid (aCSF; 124 mM NaCl, 3 mM KCl, 26 mM
NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgSO4,
and 10 mM glucose). A midsagittal cut was made in the brain
and one hemisphere was returned to ice-cold aCSF until re-
quired. Hippocampal slices were cut transversely (400 μm
thick) using a Mcilwain tissue chopper (Mickle Laboratory
Engineering Co. Ltd., UK) and stabilized for 1 h in aCSF
Mol Neurobiol
constantly perfused with a gas mixture of 95% O2 and 5%
CO2 at room temperature. For treatment, slices were trans-
ferred to a treatment chamber containing aCSF constantly
perfused with a gas mixture of 95% O2 and 5% CO2. To
examine the effect of Avn-C on Aβ42-induced LTP impair-
ment, the mouse hippocampal slices were pretreated with de-
sired concentrations of Avn-C for 0.5 h, followed by 0.5 μM
Aβ42 for another 2 h in the same aCSF medium at room
temperature. In case of transgenic mice experiments, slices
were treated with 50 μM Avn-C in the aCSF medium for
2 h at room temperature. For inhibitor studies with transgenic
mice hippocampi, slices were pretreated with antagonists for
1 h followed by 50 μM Avn-C exposure in the same aCSF
medium.
Electrophysiology
Slices were allowed approximately 60 min to recover from the
slice procedure and for stable responses to be obtained.
Extracellular field potentials were recorded in the CA1 region
using microcapillary electrodes (3–5 MΩ) containing NaCl
(3 M). Stimulating electrodes were placed in the subiculum
and CA3 (Schaffer collateral pathway). Stimuli were delivered
alternatively to the two electrodes (each electrode 0.016 Hz).
After establishing a stable baseline for 30 min, LTP was
evoked by two trains of tetanus stimuli (each 100 Hz, 1 s:
repeated after a 30-s interval), field excitatory postsynaptic
potentiation (fEPSP) was recorded for at least 60 min. The
slope of the evoked field potential response was measured
and expressed relative to the normalized preconditioning
baseline. Data were collected by an NI USB-6251 data acqui-
sition module (National Instruments, Texas, USA), amplified
by an Axopatch 200B amplifier (Axon Instruments, CA,
USA), and captured and analyzed using WinLTP software
(www.winltp.com).
Amyloid-β Preparation
The amyloid-β 1-42 peptide (Aβ42; Abcam, Cambridge, UK)
was initially dissolved at a concentration of 1 mg/mL in 100%
HFIP (1,1,1,3,3,3-hexafluoro-propanol; Sigma-Aldrich). This
solution was then incubated at room temperature for 1 h, with
occasional mixing on a vortex at a moderate speed. Next, the
solution was sonicated for 10 min in a water bath sonicator.
The HFIP/peptide solution was then dried under a gentle
stream of nitrogen gas. DMSO (100%) was used to resuspend
the peptide, which was then incubated at room temperature for
12 min with occasional mixing using a vortex. The final so-
lution was aliquoted into smaller volumes and stored at −
80 °C. Oligomeric Aβ preparation was done as described
previously [28]; 500–1000 μL of D-PBS (Invitrogen, UK)
was added to the peptide stock solution and incubated for
2 h at room temperature to allow for peptide aggregation.
Avenanthramide-C Administration
Avn-C was purchased from Sigma-Aldrich. For treatment,
final concentration of 1 mg/mL Avn-C was prepared by dis-
solving in 10% Kolliphor (Sigma-Aldrich, USA). Mice
(5XFAD or Tg2576) were divided into three groups: Tg
Avn-C-treated (n = 10), Tg vehicle-treated (n = 7), and non-
transgenic vehicle-treated (n = 9). At 6 months-of-age, Avn-
C or vehicle was administered orally to each group of mice
once a day (2, 4, and 6 mg/kg, at approximately 4:00 p.m.) for
desired days.
Morris Water Maze Test
At 2 weeks after the first Avn-C administration, mice were
subjected to the Morris water maze (5XFAD: n = 8; Avn-C-
treated 5XFAD: n = 10; WT: n = 8). Mice were placed in a
circular pool (114 cm in diameter, 25 cm in height) filled with
water that was maintained at 25 °C and made opaque using
nontoxic paint. A fixed-position, clear escape platform (17 ×
10.5 cm) was submerged 1.5 cm below the surface of the
water for all training sessions. In each trial, mice were ran-
domly placed facing the wall in any of the three quadrants of
the pool that did not contain the platform. A total of 16 trials
(four trials per day for four consecutive days) were performed.
Mice were allowed to swim for 40 s. Mice that did not find the
platform within 40 s were guided to it. The animals were
allowed to stay on the platform for 10 s. A probe test was
performed without the platform in place 24 h after the last
training trial. In the probe test, the mice were allowed to swim
for 60 s to search for the removed escape platform.
CSF Analysis
Cerebrospinal fluid (CSF) collection was always performed
between 10 a.m. and 2 p.m. Mice were injected with a mixture
of 10 IU ketamine and Rompun in 3:1 ratio and placed in a
chamber until they were deeply anesthetized. CSF was then
immediately collected from cisterna magna with the help of
magnification glasses. To this end, the overlying skin was
incised to expose the skull and the posterior neck muscles.
The latter were cut off layer by layer until the cisterna magna
was visible through the translucent dura matter. After cleaning
any blood residue from the surface with a cotton swab, the
dura was perforated with a capillary tube and CSF was col-
lected. Then the capillary tube was connected with a 3-mL
syringe through a polyethylene tubing that has a 1-mm inter-
nal diameter and the collected CSF was transferred in to a pre-
marked polypropylene tubes and immediately placed on dry
ice before stored to − 80 °C freezer for further use. Typically, a
total of 15 to 20 μL of CSF was collected.
 Mol Neurobiol

Open Field Test explored the objects for 10 min. The percentage of preference
index was calculated as the time exploring the novel object (n)
At 3 weeks after the first Avn-C administration, mice were divided by the time exploring both novel and familiar objects
subjected to behavior tests (5XFAD: n = 8; Avn-C-treated (n + f) [% preference index = n/(n + f) × 100].
5XFAD: n = 10; WT: n = 9). Locomotor activity was mea-
sured in open field test as described [29]. Mice were placed
Immunohistochemistry
in the center of the arena (40 × 40 × 40 cm). Mice were pre-
habituated in arena for 5 min and then freely explored arena
Wild-type (WT) and transgenic mice (5XFAD and Tg2576)
for 20 min. Horizontal movement of mice in arena was re-
treated with vehicle or Avn-C (n = 3) were euthanized after
corded and total distance that mice moved in the arena were
2 weeks of vehicle or drug treatment. The brain was isolated,
measured by ANY-maze (Stoelting, IL, USA).
fixed in 4% paraformaldehyde, and transferred to 30% sucrose
overnight to prevent tissue damage. The tissues were embed-
Rotarod
ded in OCT compound and sliced to a thickness of 20 μm
using a Leica CM1860 tissue processor (Leica Biosystems,
Mice were placed on the stationary cylinder of rotarod
Germany). Brain sections were mounted onto glass slides
(5XFAD: n = 8; Avn-C-treated 5XFAD: n = 10; WT: n = 8)
and permeabilized with 0.3% Triton X-100 for 1 h. After
and the test was performed as described [30]. During pre-
washing with Tris-buffered saline, the sections are incubated
training, mice were habituated on the cylinder (B.S.
in 5% BSA. Primary antibodies for anti IBA1 (1:1000, Wako)
Technolab INC., Seoul, Korea) rotating at a constant speed
and anti-β-amyloid (6E10, 1:500, BioLegend) were applied to
(4 rpm for 60 s). Once the mice were able to stay on the
the samples overnight at 4 °C. After being washed with PBS,
cylinder for 60 s, test was performed 3 h later. After placing
sections were probed with FITC- or TRITC- conjugated sec-
the animal on the cylinder, rotating speed was accelerated
ondary antibodies (1:500) at room temperature for 2 h, follow-
from 4 to 40 rpm over 5 min. Four trials were performed
ed by washing with PBS. Slides were mounted with anti-fade
and minimum 5 min breaks were given between trials. Time
mounting media. Confocal images were obtained using an
to fall from the cylinder was recorded with a cutoff time of
LSM 520 confocal microscope (Carl Zeiss).
300 s. Total eight trials were performed and the data from the
For analysis of the amyloid aggregates, the ImageJ soft-
last trials were averaged.
ware (https://rsbweb.nih.gov/ij/) was used. After adjusting
for threshold, ImageJ was used to measure total area of the
Pole Test
plaques and the percentage of total brain area occupied by
plaques. Data were pooled from 3~5 sections at ×200
Pole test was performed as described [31]. A vertical pole with
magnification of each mouse and three mice were used for
a rough surface (1.5 cm diameter, 43 cm height) was placed in
the statistical analysis. For quantification of Iba-1, 3 coronal
the home cage. The mice were placed facing upward on the
sections per mouse were selected. Optical density was
top of a pole (5XFAD: n = 8; Avn-C-treated 5XFAD: n = 10;
determined with ImageJ software. Interested cells have been
WT: n = 8). The mice were monitored for 60 s or until they
selected, and area integrated intensity and mean gray value
arrive at the bottom. The pole test score was calculated as
have been measured. Finally, the total cell fluorescence
follows: fell from the pole immediately: 1; fell from the pole
calculated. The number of animals in each group was
in 0–10 s: 2, 11–20 s: 3, 21–30 s: 4, 31–40 s: 5, 41–50 s: 6, 51–
indicated in the figure legends.
60 s: 7; stayed on the pole for 60 s and climbed halfway down:
8; climbed to lower half of pole: 9; and climbed down and off
in: 51–60 s: 10, 41–50 s: 11, 31–40 s: 12, 21–30 s: 13, 11– Sandwich Enzyme-Linked Immunosorbent Assay
20 s: 14, 1–10 s: 15.
Quantitative determination of pro-inflammatory cytokines
Novel Object Recognition Test TNFα, IL-6, and IL-10 was performed using the Mouse
(TNF alpha, IL-6, and IL-10) enzyme-linked immunosorbent
Mice were subjected to the novel object recognition test assay kit (eBioscience, San Diego, USA) according to manu-
(5XFAD: n = 8; Avn-C-treated 5XFAD: n = 10; WT: n = 9). facturer’s instructions. Tissue lysate was prepared from the hip-
The test consists of two exploration phase: training and test pocampus using tissue extracting reagent I (Thermo Scientific
phase. During the training phase, mice were placed in the FNN0071). Mouse TNF alpha ELISA Ready-SET-Go!® kit
open-top arena (40 × 40 × 40 cm) for 10 min in the presence (colorimetric; eBioscience; sensitivity 4 pg/mL; range 4–
of two identical objects. In the test phase, mice were returned 500 pg/mL), Mouse IL-6 alpha ELISA Ready-SET-Go!® kit
to the arena with one novel object and the other object which (colorimetric; sensitivity 8 pg/mL; range 8–1000 pg/mL;
is identical to the one used in the training phase and then eBioscience), and Mouse IL-10 alpha ELISA Ready-SET-
Mol Neurobiol
Go!® kit (colorimetric; sensitivity 8 pg/mL; range 8–1000 pg/
mL; eBioscience) were used according to following protocol.
ELISA plate coated with 100 μL/well of capture antibody
in coating buffer and the sealed plate incubated overnight at
4 °C. The wells aspirated and washed three times with >
250 μL/well wash buffer. Allowing time for soaking (~
1 min) during each wash step increases the effectiveness of
the washes. The plate was blotted on absorbent paper to re-
move any residual buffer. The wells blocked with 200 μL/well
of 1× ELISA/ELISPOT diluent at room temperature for 1 h.
One hundred microliters of top standard concentration was
added to the appropriate wells. Twofold serial dilutions of
the top standards performed to make the standard curve for a
total of 8 points. Then 100 μL of samples added to the appro-
priate wells. The sealed plate incubates at room temperature
for 2 h (or overnight at 4 °C for maximal sensitivity). The
wells aspirated and washed three to five times. Detection an-
tibody diluted in 1× ELISA/ELISPOT diluent and 100 μL
added to each well and allowed to incubate at room tempera-
ture for 1 h. Again, the wells aspirated and washed three to
five times. Each well was added with 100 μL of Avidin-HRP
diluted in 1× ELISA/ELISPOT diluent and incubated at room
temperature for 30 min. One hundred microliters of 1× TMB
Solution was added to each well followed by five to seven
washes. After the 15 min of incubation, 50 μL of Stop
Solution was added to each well. The plate was read at
450 nm.
Immunoblotting
Hippocampal tissue lysates were prepared using 1× RIPA
Buffer (Cell Biolabs, Inc., Catalog number: AKR 190) con-
taining protease inhibitor cocktail (immediately added before
use). Protein concentrations were determined in a BCA assay.
Proteins were separated in 10–12% gels and transferred to
PVDF membranes (Millipore, Bedford, MA, USA), which
were incubated overnight with primary antibodies. The fol-
lowing primary antibodies were purchased from Cell
Signaling Technology (MA, USA): anti-caspase-3 (1:1000
dilution), anti-cleaved caspase-3 (1:500), anti-NF-κB-p65
(1:1000), anti-phospho-NF-κB-p65 (1:1000), anti-phospho-
IKKα/β (1:1000), anti-GSK-3α/β (1:1000), anti-phospho-
GSK-3β(Ser9) (1:1000), and anti-β-actin (1:1000). The anti-
IKKα/β (1:1000) antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Membranes were
then incubated with rabbit IgG antibodies (1:5000, Cell
Signaling Technology) conjugated to horseradish peroxidase
and immunoblotted using an ECL detection system
(Millipore, Bedford, MA, USA). Densitometric analysis of
the immunoreactive bands was carried out using ImageJ soft-
ware (National Institutes of Health, Bethesda, MD). Intensity
of bands from the protein of interest were normalized to the
intensity of actin bands of the respective blots. The statistical
significance of the data was analyzed by Student’s t test. A P
value < 0.05 was considered statistically significant.
Biotinylation and Streptavidin Pull-Down Assays
Biotinylation of Avn-C was performed according to the man-
ufacturer’s G-Biosciences (catalog number: BS-16, BS-17)
instructions. For pull-down experiments, slices were treated
with biotinylated Avn-C (50 μM final concentration) in the
presence or absence of prazosin hydrochloride. After treat-
ment, tissue was homogenized in lysis buffer containing
25 mM Tris (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium
deoxycholate, 0.1% SDS, 1 mM NaF, and a cocktail of prote-
ase inhibitors (Sigma). The lysate was centrifuged at 11,000×g
for 15 min at 4 °C to remove nuclei and cellular debris. Total
protein concentration was determined in a BCA assay
(Pierce). A small amount of the lysate was removed for later
whole-cell analysis. Subsequently, 100 μL of streptavidin
beads (Thermo Scientific Inc.) was added to 600 μg of protein
lysate and placed on a rotator at 4 °C for 2 h. Samples were
then washed five times in wash buffer (25 mM Tris pH 7.6,
150 nM NaCl, 0.5% Triton X-100); beads were pulled down
after each wash by gentle centrifugation. Bound proteins were
eluted in 2× SDS reducing buffer and gently heated at 60 °C
for 30 min. The resulting supernatant was transferred to new
tubes and heated at 90 °C for 5 min before gel loading.
Statistical Analysis
All data are means ± s.e.m. of at least three independent ex-
periments. Statistical analyses were performed with two-
tailed, unpaired Student’s t test. Values of P > 0.05 were con-
sidered not significant (n.s.); values of P < 0.05 and P < 0.01
were considered significant and highly significant,
respectively.
Results
Avenanthramide-C Restores Aβ-Mediated LTP
Inhibition and Impaired LTP in AD Mouse Models
To analyze the protective effects of Avn-C on Aβ-mediated
impairment of synaptic function, we conducted LTP
analysis—a technique used to study synaptic events occurring
during learning and memory. We treated slices of WT mouse
hippocampi (an acute preparation) with Aβ42 oligomers in the
presence or absence of different concentrations of Avn-C (10,
25, and 50 μM) and analyzed LTP. Slices treated with Aβ42
(0.5 μM, 2 h) alone showed impaired LTP, whereas transient
pretreatment with Avn-C (50 μM, 30 min) followed by co-
treatment with Aβ42 restored LTP (Fig. 1a). Avn-C at 10 and
25 μM concentrations did not improve the LTP significantly

Fig. 1 Avn-C restores Aβ-mediated LTP inhibition and cognitive deficits.
a High-frequency stimulation (2× tetanus) induces LTP in control slices
(closed circles, 158 ± 5%, n = 6), but not following Aβ treatment (open
circles, 102 ± 7%, n = 6). However, Avn-C pretreatment blocks Aβ-
mediated LTP inhibition (triangles, 152 ± 10%, n = 6); P < 0.05. b Aβ
treatment alters the levels of GSK3β (Ser9) and caspase-3 (C-3, cleaved
C-3; Clv. C-3) active forms, whereas Avn-C pretreatment normalizes these
protein levels (n = 3). c LTP was induced in hippocampal slices from WT
(open circles, 150.8 ± 3%, n = 5) and Avn-C pretreated 5XFAD mice (tri-
angles, 135 ± 3%, n = 5) but not in 5XFAD controls (closed circles, 112 ±
Mol Neurobiol
4%, n = 5); P < 0.01. d Levels of active forms of GSK3β (Ser9) and
caspase-3 in WT, 5XFAD control, and Avn-C-treated hippocampal slices
(n = 3). e LTP is induced in hippocampal slices from another
amyloidogenic mouse model Tg2576 mice pretreated with Avn-C (trian-
gles, 138 ± 3%, n = 5) and WT (open circles, 151.2 ± 4%, n = 5) but not in
Tg2576 control (closed circles, 105 ± 3%, n = 5); P < 0.05. f Levels of
GSK3β (Ser9) and caspase-3 active forms in WT, Tg2576 control, and
Avn-C-treated hippocampal slices (n = 3). In all panels, error bars indicate
the SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Black box in LTP data
indicates tetanus stimulation
Mol Neurobiol
(Additional file 1: Fig. S1a, b). Involvement of caspase-3 and
GSK3β in Aβ42-mediated LTP impairment has been reported
[7]. To confirm whether Avn-C exerts its effects by inhibiting
these molecules, we performed immunoblot analyses to detect
activity of GSK3β and caspase-3 in hippocampal slices; con-
sistent with previous reports we found increased cleaved
caspase-3 levels and decreased phospho-GSK3β (Ser9)
levels, in Aβ42-treated samples (Fig. 1b) [7, 32]; however,
50 μM Avn-C treatment restored these to basal levels. Also,
we investigated 50 μM Avn-C’s effect on WT mouse hippo-
campal LTP and the activity of GSK3β and caspase-3. LTP
analysis and immunoblot results revealed that Avn-C has no
effect on WT mouse hippocampal LTP (Additional file 2: Fig.
S2a, b).
To assess the effects of Avn-C on synaptic function in a
transgenic mouse model of AD, we treated hippocampal slices
from 5- to 6-month-old 5XFAD mice ex vivo with Avn-C, and
performed LTP. LTP generated in the 5XFAD mice slice was
significantly reduced compared with the WT controls.
Remarkably, Avn-C treatment significantly increased the
fEPSP slope of the 5XFAD mice hippocampal slices com-
pared with that of the untreated controls (Fig. 1c). Consistent
with previous results (Fig. 1b), Avn-C treatment (50 μM) re-
duced cleaved caspase-3 levels and increased p-GSK3β
(Ser9) levels, comparable with wild-type controls (Fig. 1d).
We also tested the effects of Avn-C on Tg2576 mice, another
mouse model of amyloid deposition. As expected, tetanus
stimulation induced LTP in WT mouse slices, but failed to
induce LTP in Tg2576 control mouse hippocampal slices.
However, Avn-C treatment (50 μM) for 2 h restored LTP in
hippocampal slices from 7- to 8-month-old Tg2576 mice (Fig.
1e). We also observed reduced cleaved caspase-3 and in-
creased p-GSK3β (Ser9) levels, in Avn-C-treated samples
(Fig. 1f). Thus, Avn-C restores hippocampal LTP irrespective
of Aβ burden in two different mouse models of AD.
Avn-C Oral Administration Improves Spatial
and Recognition Memory in AD Transgenic Mice
Next, we examined the effects of Avn-C in vivo. 5XFAD mice
aged 5–6 months were treated with Avn-C (2, 4, and
6.0 mg/kg, oral administration) or vehicle once daily for 1 or
2 weeks and brain slices were prepared from these mice and
analyzed for hippocampal LTP ex vivo (Fig. 2a). One week
Avn-C treatment did not improve impaired LTP at any given
dosages in 5XFAD mice (data not shown). In contrast, 2 weeks
Avn-C treatment at 6 mg/kg was associated with increased
LTP (Fig. 2d), suppressed cleaved caspase-3, and increased
p-GSK3β (Ser9) levels comparable to the vehicle-treated con-
trols (Fig. 2e), whereas 2 mg/kg Avn-C treatment showed no
effect in LTP while 4 mg/kg showed little, but not significant
enhancement in impaired LTP in 5XFAD mice (data not
shown). Following this, similar LTP and immunoblot results
were observed in (6.0 mg/kg) Avn-C-treated 7–8 months aged
Tg2576 mice (Fig. 2b, c).
Based on these observations, we decided to check whether
the oral treatment with Avn-C could improve cognition in AD
mice. To do that, we orally administered 5XFAD mice (5–
6 months old) with Avn-C (6 mg/kg) or vehicle once daily
for 2 weeks. At the end of the treatment, CSF was collected
from 5XFAD mice and analyzed for the presence of Avn-C.
CSF analysis confirmed that Avn-C can cross through the
blood-brain barrier (Additional file 3: Fig. S3). Avn-C treat-
ment improved novel object recognition memory in 5XFAD
mice comparable with the wild-type counterpart while the
vehicle-treated 5XFAD mice scored poorly (Fig. 2f). Next
we employed Morris water maze (MWM) test—a spatial
memory task that depends on the hippocampus, to analyze
whether Avn-C could enhance learning and memory retrieval.
The mice were trained on the fixed hidden platform with vi-
sual reference. The platform was removed at 24 h after the last
training and the mice were tested to visit the location of re-
moved platform. Compared to WT mice, vehicle-treated
5XFAD mice were severely impaired in the probe test
displaying significant loss of the memory for the platform
location whereas Avn-C treated 5XFAD mice showed less
latency time in seconds to reach the hidden platform and sig-
nificantly increased time spent in the target quadrant with
increased number of platform crossings indicating rescue of
reference memory (Fig. 2g–j). We also tested motor-related
behavioral phenotypes, including rotarod test, pole test, and
open field test, in 5XFAD mice after Avn-C treatment and
observed no significant effect in basal motor function
(Additional file 4: Fig. S4a–c). Collectively, these data suggest
that Avn-C treatment restores impaired synaptic function
in vivo in two different mouse models of AD pathology and
also indicate that at least 2 weeks of Avn-C (daily dosage,
6 mg/kg) treatment is necessary to reverse the impaired cog-
nition in AD mice.
Avn-C Administration Suppresses Neuroinflammation
But Had No Effect on Aβ Pathology
Following these results, we determined to check whether
this behavioral improvement could be the effect of Avn-C
on Aβ pathology in AD mice. For that, we assessed the
Aβ burden in these Avn-C-treated AD transgenic mice
hippocampi. Immunoblot analysis showed that 2 weeks
Avn-C treatment did not have any effect on hippocampal
Aβ-related protein expression. No significant difference
was found in the protein levels of BACE1, APP, RIPA-
soluble Aβ fractions, and phospho-tau between vehicle-
a n d Av n - C - t r e a t e d t r a n s g en i c m i c e h i p p o c a m p i
(Fig. 3a, b). 5XFAD mice begin accumulating amyloid
depositions as early as 2 months of age and increasing
rapidly with age. We next performed Aβ histopathology
Mol Neurobiol
 Mol Neurobiol
ƒFig. 2 Avn-C oral administration improves cognitive deficits in AD
mice. a Avn-C or vehicle was orally administered for 2 weeks (6 mg/kg
bodyweight per day), followed by analysis of LTP, recognition and spatial
learning, and memory tasks. b–e Two weeks Avn-C oral treatment res-
cued LTP (Tg2576 + Avn-C—open circles, 154.6 ± 6%, n = 6; 5XFAD +
Avn-C—open circles, 151.8 ± 3%, n = 6) and restored GSK3β (Ser9) and
caspase-3 active forms to their basal level in both mice hippocampal
slices. f Avn-C ameliorates impaired object recognition memory in
5XFAD mice. Bar diagram indicates the object preference index
(5XFAD: n = 8; Avn-C-treated 5XFAD: n = 10; WT: n = 8). f (top)
Schematic representation of novel object recognition test. g–j Avn-C also
restored impaired spatial memory during the Morris water maze in
5XFAD mice (5XFAD: n = 8; Avn-C-treated 5XFAD: n = 10; WT: n =
8). g (top) The apparatus setting for Morris water maze. The square
indicated in the southeast quadrant (3) represents the site of the hidden
platform. Mice were placed into the northwest quadrant to start each trial
(1). g (below) The swim paths of representative individual mice in the last
training trial. h Latency to hidden platform in the training session. Error
bar indicates the SEM; *P < 0.05, **P < 0.01, ***P < 0.005, significant
between wild type and 5XFAD. #P < 0.05, ##P < 0.01, ###P < 0.005,
significant between 5XFAD and 5XFAD-Avn-C. i Time spent in the
target quadrant in the probe test. j Number of mice which crossed the
location of (removed) platform in probe test. In all panels, error bars
indicate the SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Black box sym-
bol in LTP data indicates tetanus stimulation
analyses in Avn-C-treated 5XFAD mice. IHC quantitative
analysis revealed no significant differences in the total
amyloid burden in the hippocampus between vehicle-
and Avn-C-treated 5XFAD mice (Fig. 3c). Growing evi-
dence from various studies has implicated the role of in-
flammation in AD progression. Increased levels of
microglial activation and pro-inflammatory markers have
been observed in brain samples from both AD patients
and AD transgenic mice models. In Tg2576 and 5XFAD
mice, a 2-week treatment with Avn-C significantly re-
duced the inflammation which was evident from reduced
levels of ionized calcium binding adapter molecule 1
(Iba1), a marker of activated microglia and inflammation.
Increased Iba1-positive microglia in the hippocampus of
vehicle-treated Tg2576 and 5XFAD mice compared to
vehicle-treated WT mice were normalized in Avn-C-
treated Tg2576 and 5XFAD mice, indicating that Avn-C
reversed inflammation (Fig. 3d, e). In addition, Avn-C
treatment significantly suppressed the levels of pro-
inflammatory cytokines TNFα and IL6 in 5XFAD and
Tg2576 mice hippocampi whereas increasing the levels
of anti-inflammatory marker IL-10 as measured by
ELISA (Fig. 3f, h). Avn-C treatment also decreased the
levels of phospho-forms of IKK and P65 which are the
major downstream molecules of cytokine mediated in-
flammatory pathways in 5XFAD and Tg2576 mice hippo-
campi (Fig. 3g, i). Taken together, these findings indicated
that Avn-C-induced memory enhancement in these mice
was independent of Aβ pathology whereas reducing pro-
inflammatory microglial activation.
Avn-C Improves Memory and Suppresses
Neuroinflammation in AD Mice Through AMPK
Activation
Polyphenolic compounds from natural products exert benefi-
cial effects by activating adenosine monophosphate-activated
protein kinase (AMPK) [33], a heterotrimeric serine/threonine
kinase that plays a critical role in cell death and survival.
Critically, AMPK inhibits inflammation by regulating several
pathways [34], and AMPK activation alleviates caspase-3 ac-
tivity by increasing expression of anti-apoptotic proteins Bcl-2
and survivin [35]. Therefore, we investigated whether Avn-C
exerts its anti-caspase-3 and anti-inflammatory effects through
AMPK activation. Immunoblotting (Fig. 4a, b) results showed
that Avn-C oral treatment (6 mg/kg, once daily for 2 weeks)
increased phospho-AMPK levels in both 5XFAD and Tg2576
mouse hippocampus. A previous study showed that AMPK
activation reduces neuropathological deficits and improves
plasticity in a mouse model of AD [36]. Our results also
showed that Avn-C restored LTP in mouse models of AD
through AMPK activation. To confirm this, we pretreated
5XFAD and Tg2576 mouse acute hippocampal slices with
the AMPK inhibitor Compound C and analyzed LTP.
Pretreatment with Compound C followed by Avn-C co-treat-
ment inhibited the effects of Avn-C on hippocampal LTP in
both 5XFAD (Fig. 4c) and Tg2576 mice (Fig. 4e), and re-
versed Avn-C-mediated increases in p-GSK3β (Ser9) levels,
along with reduced cleaved caspase-3 levels, in both models
(Fig. 4d–f). Compound C alone did not have any impact on
control LTP in both AD mice (Additional file 5: Fig. S5a, b).
Avn-C Induces AMPK Activation Through α-1A
Adrenergic Receptors
Brain AMPK activation is mainly regulated by α-adrenergic
receptors (α-AR). On ligand binding, α-ARs activate liver ki-
nase B1 or calcium/calmodulin-dependent kinase kinase or in-
crease AMP levels, leading to subsequent phosphorylation and
activation of AMPK [37, 38]. α-1A adrenergic receptors are
generally stimulatory, upon their ligand binding showed to pro-
mote hippocampal synaptic plasticity [39]. Phenylephrine is a
commercially available selective agonist of α1A adrenergic re-
ceptor (α-1A AR) and proven to regulate AMPK by phosphor-
ylating at two different sites (Thr 172-activation or Ser 485/491-
inhibition) [40]. Apart from these two ligands, Leptin is a hor-
mone that can bind and activate α1A AR. Leptin binding of α-
1A AR leads to AMPK phosphorylation at Thr 172 and activa-
tion [41, 42]. Moreover, leptin was also strongly reported to
enhance hippocampal LTP [43] and AMPK activation [44].
Based on these observations from different studies, we speculate
the possibility that Avn-C also may execute its LTP-restoring
effect through interacting with α-1A AR similar to that of leptin.
To test this, we used prazosin, an irreversible α-1A AR inhibitor
 Mol Neurobiol

a b c d
Tg2576 5XFAD
Tg2576 - Vehicle 5XFAD - Vehicle 5XFAD - Avn-C
Veh Avn-C Veh Avn-C 5XFAD - Vehicle

Normalized O.D. (% of control)

Tg2576 - Avn-C 5XFAD - Avn-C Vehicle

Flourescence intesnsity (%)

Normalized O.D. (% of Control)
BACE1 CA1 CA1 Avn-C
BACE1 n.s. n.s. n.s.
120 n.s n.s n.s n.s n.s.
120 100 n.s
APP 100 APP 100
80

6E10
80 80
60
60 60
40 40
40
p - Tau p-Tau 20 20
20
0 0
0

p-
BA

AP
p-
-actin
AP
BA

-actin

Ta
CE

P
Ta
P

u
CE

1
1

e f WT - Vehicle g h WT - Vehicle
Tg - Vehicle 5X - Vehicle
5X - Avn-C

Fluorescence intesnsity (%)

Tg - Avn-C

Fluorescence intesnsity (%)

WT - Vehicle Tg2576 - Vehicle Tg2576- Avn-C WT - Vehicle 5XFAD - Vehicle 5XFAD - Avn-C
150 150 ** ***
* **
Iba-1

100

Iba-1
100

Iba-1
50 50

0 0

i WT - Vehicle j k WT - Vehicle l
200
Normalized O.D. (% of control)

Tg2576 - Vehicle 5XFAD - Vehicle

150

Normalized O.D. (% of control)

Tg2576 - Avn-C ** 5XFAD - Avn-C 200
Tg2576 100 5XFAD
150 ***
Veh Avn-C Veh Avn-C
50 100
Conccentration (pg/ml)
100 150 ** ** 120 *** *** 150
Conccentration (pg/ml)

100 *** * 100

0 *** *** 50
80 * * 80 125 80 100 125 0
100 200 80 100
60 60 * * p65 60 200
75 150 60 75 p65
40 40 *** 40 150 ***
50 p-p65 100 40 50 p-p65 100
20 20 25 50 20 20
-actin 25 50
-actin
0 0 0 0 0 0 0 0
IL-6 IL-10 p65 p-p65 IL-6 IL-10 p65 p-p65

Fig. 3 Avn-C did not reduce amyloid burden but reduced than in those from vehicle-treated controls (scale bar; 20 μm). h
neuroinflammation in AD mice. a, b Western blot and quantitative Quantitative analyses of fluoroscence intensities are shown in graphs. i
analysis of BACE1, APP, Aβ, and phopho-tau levels in hippocampal Enzyme-linked immunosorbent assay (ELISA) histograms (relative) of
extracts from vehicle (Veh) or Avn-C treated (2 weeks) Tg2576 and IL-6, TNFα, and IL-10 in hippocampal homogenates of WT and Tg2576
5XFAD mice. The error bars indicate the mean ± s.e.m. (n = 4). c mice treated with either Avn-C or vehicle. All of the methods and proce-
Fluorescence images of hippocampal regions of vehicle- or Avn-C treated dures recommended by the manufacturer were followed, and experiments
(2 weeks) 5XFAD mice, indicating the localization of Aβ (6E10) aggre- were performed in triplicate. j Western blot and qauntitative analysis of
gates (scale bar; 200 μm). d Quantitative analyses of fluoroscence inten- IKK, p65, and their active form levels in hippocampal extracts from
sities are shown in graphs. e Immunohistological analysis shows reduced vehicle- (Veh) or Avn-C-treated (2 weeks) Tg2576 mice. k ELISA histo-
levels of microglial activation comparable with WT mouse hippocampus grams (relative) of IL-6, TNFα, and IL-10 in hippocampal homogenates
in Avn-C-treated Tg2576 mouse hippocampal slices than in those from of WT and 5XFAD mice treated with Avn-C or vehicle. l Western blot and
vehicle-treated controls (scale bar; 20 μm). f Quantitative analyses of quantitative analysis of IKK, p65, and their active form levels in hippo-
fluoroscence intensities are shown in graphs. g Immunohistological anal- campal extracts from vehicle- (Veh) or Avn-C-treated (2 weeks) 5XFAD
ysis shows reduced levels of microglial activation comparable with WT mice. In all panels, error bars indicate the SEM; *P < 0.05, **P < 0.01,
mouse hippocampus in Avn-C-treated 5XFAD mouse hippocampal slices ***P < 0.001

[45]. Pretreatment with prazosin blocked the effect of Avn-C on activation of caspase-3 and pro-inflammatory markers while
hippocampal LTP in both 5XFAD (Fig. 5a) and Tg2576 (Fig. increased GSK3β (Ser9) phosphorylation and anti-
5c) mice, and reversed the Avn-C-mediated increases in p- inflammatory marker (Fig. 6).
AMPK and p-GSK3β (Ser9) levels, as well as reduced cleaved
caspase-3 levels (Fig. 5b, d). Prazosin alone did not have any
impact on control LTP in both AD mice (Additional file 5: Fig.
S5c, d). To confirm Avn-C binding to α-ARs, we pretreated Discussion
mouse hippocampal slices with biotinylated Avn-C in the pres-
ence or absence of prazosin. Homogenized hippocampal lysates Here we report for the first time that Avn-C, an oats
were pulled down with streptavidin beads and the bead fractions component, restored impaired memory, a major trait in
immunoblotted for α-1A ARs. The pull-down results confirmed AD condition, in both ex vivo and in vivo studies. Avn-
that biotinylated Avn-C binds to α-1A AR (Fig. 5e). Together, C alleviated AD-related memory impairment by
our results demonstrate that Avn-C rescued Aβ-mediated mem- strengthening synapses which was evident from en-
ory impairment via α-1A AR and AMPK results in reduced hanced LTP, reducing caspase-3 and pro-inflammatory
markers activation, and increasing pS9GSK3β and
Mol Neurobiol

a WT b WT
5XFAD Tg2576
WT 5XFAD 5XFAD + Avn-C WT Tg2576 Tg2576 + Avn-C
__ __ + __ __ +

Normalized O.D.

Normalized O.D.
Avn-C 200 Avn-C 200

(% of control)

(% of control)
AMPK 150 * ** AMPK 150
* **
100 100
p -AMPK p -AMPK
50 50
β-actin 0 β-actin 0
AMPK p-AMPK AMPK p-AMPK

c 1 2 2+3 5 6 5+6 d

1 mV
Con Avn-C
3 4 3+4 Compound C + Avn-C
10 ms 200

Normalized O.D. (% of control)

Compound C (μM) 0 0 10 150
350 ** **
Avn-C (μM) 0 50 50
Normalized fEPSP slope

Control 100
300 Avn-C C-3 50
(%) of baseline)

Comp.C + Avn-C Clv . C -3 0

250 C-3 Clv. C-3
GSK-3ɑβ 200
200 4 ** *
150
pS9GSK-3β
150 6 100
1 5
100 2
E β-actin 50
3 5X FAD 0
50 GSK-3ɑβ pS9GSK-3β
0 10 20 30 40 50 60 70 80 90 100
Time (min)

e 1 2 1+2 5 6 5+6 f
Con Avn-C
1 mV

3 4 3+4 Compound C + Avn-C

200
10 ms
Normalized O.D. (% of control)
Compound C (μM) 0 0 10 150 * *
350 Avn-C (μM) 0 50 50
Control 100
300 Avn-C C-3 50
Normalized fEPSP slope

Comp.C + Avn-C 0
Clv . C -3
(%) of baseline)

250 C-3 Clv. C-3

200
150
1 5
100
3 Tg2576
50
0 10 20 30 40 50
Time (min)
Fig. 4 Avn-C restores impaired LTP by inducing AMPK activation. a, b
Immunoblot validation and densitometry analysis of (n = 3) AMPK and
phospho-AMPK from WT, vehicle-, or Avn-C-treated 5XFAD and
Tg2576 mouse hippocampal samples. c Acute hippocampal slices from
5XFAD mice were pretreated with the AMPK inhibitor Compound C
(10 μM) for 1 h prior to Avn-C (2 h) exposure. Preincubation with
Compound C blocks the effect of Avn-C on LTP (control, n = 5, 117.6
± 3%, closed circles; Avn-C, 163.8 ± 5%, n = 5, open circles; Compound
C + Avn-C, 124.1 ± 8%, n = 4, triangles; P < 0.05. d Immunoblot valida-
tion and densitometry analysis of GSK3β (Ser9) and caspase-3 active
GSK-3ɑβ 200
4 * *
150
pS9GSK-3β
6 100
β-actin
2 50
0
GSK-3ɑβ pS9GSK-3β
60 70 80 90 100
forms in Compound-C- and Avn-C-treated 5XFAD mouse hippocampi
(n = 3). e Preincubation of Tg2576 mouse acute hippocampal slices with
Compound C blocks the effect of Avn-C on LTP (control, 119.5 ± 2.5%,
n = 5 closed circles; Avn-C, 160.8 ± 5.8%, n = 4, open circles; Compound
C + Avn-C, 122.7 ± 2.3%, n = 4, triangles; P < 0.05). f Immunoblot vali-
dation and densitometry analysis (n = 3) of caspase-3, GSK3β (Ser9)
active forms in compound-C- and Avn-C-treated Tg2576 mouse acute
hippocampal samples. Error bars indicate the SEM. *P < 0.05;
**P < 0.01. Black box symbol in LTP data indicates tetanus stimulation
 Mol Neurobiol

a b
1 2 1+2 3 4 3+4 Con Avn-C

1 mV
Prazosin.HCL+ Avn-C

5 6 5+6 10 ms 200 ** * 200

Prazosin.HCL - - +

Normalized O.D. (% of control)

- + + 150 150 ** **
Avn-C
350 100
Normalized fEPSP slope

Control AMPK 100

300 Avn-C 50 50
(%) of baseline)

Prazosin.HCL + Avn-C p-AMPK

0 0
250 AMPK p-AMPK C-3 Clv. C-3
C-3
200 200
4 Clv . C -3 * *
150
150
1 5 6 GSK-3ɑβ 100
100
2 pS9GSK-3β 50
3 5X FAD
50 0
β-actin GSK-3ɑβ pS9GSK-3β
0 10 20 30 40 50 60 70 80 90 100
Time (min)

c d
1 2 1+2 5 6 5+6 Con Avn-C
1 mV

Prazosin.HCL+ Avn-C

10 ms
3 4 3+4 Prazosin.HCL - - + 200 * 200
**

Normalized O.D. (% of control)

Avn-C - + + 150 150
100 100
Control AMPK
300
Normalized fEPSP slope

Avn-C 50 50
Prazosin.HCL + Avn-C p-AMPK
0 0
(%) of baseline)

250
AMPK p-AMPK C-3 Clv. C-3
C-3
200 200
4 Clv . C -3 *
150 150
6
1 5 GSK-3ɑβ 100
100 2
3 pS9GSK-3β 50
Tg2576
50 0
β-actin GSK-3ɑβ pS9GSK-3β
0 10 20 30 40 50 60 70 80 90 100

Time (min)

e Input
Beads
(5%)

α1a- AR

β -actin

Avn-C
Biotin-Avn-C
Prazosin+ Avn-C
Fig. 5 Avn-C restores impaired LTP by inducing AMPK activation
through α-1A adrenergic receptors. a Prazosin pretreatment of hippocam-
pal slices from 5XFAD mice blocks the effect of Avn-C on LTP (control,
122.3 ± 4%, n = 4, closed circles; Avn-C, 164.0 ± 7%, n = 4, triangles;
Prazosin + Avn-C, 125.8 ± 3%, n = 4, open circles; P < 0.05). b
Immunoblot validation and densitometry analysis of western blot bands
of (n = 3) AMPK, GSK3β (Ser9), and caspase-3 active forms in prazosin-
and Avn-C-treated 5XFAD mouse hippocampus. c Prazosin hydrochlo-
ride pretreatment of Tg2576 mouse hippocampal slices blocks the effect
of Avn-C on LTP (control, 116.3 ± 0.8%, n = 6, open circles; Avn-C,
- + - -
- - + +
- - - +
155.5 ± 3.6%, n = 6, triangles; prazosin + Avn-C, 116.8 ± 3.2%, n = 5,
closed circles; P < 0.05). d Immunoblot validation and densitometry anal-
ysis of (n = 3) of AMPK, GSK3β (Ser9), and caspase-3 active forms in
prazosin- and Avn-C-treated Tg2576 mouse hippocampus. Error bars
indicate the SEM. *P < 0.05; **P < 0.01. Black box symbol in LTP data
indicates tetanus stimulation. e Avn-C binds α-1A adrenergic receptors
(α-1A AR). Hippocampal slices were treated with biotinylated Avn-C in
the presence or absence of prazosin. Tissue lysates were pulled down with
streptavidin beads, followed by western blotting for α-1A AR. In all
panels, error bars indicate the SEM; *P < 0.05; **P < 0.01; ***P < 0.001

IL10 levels in two different mouse models of AD. Our molecules in AD and inhibitory effect of Avn-C on
data support the generally adverse role of these these molecules results in memory restoration. The
Mol Neurobiol
Fig. 6 Schematic representation of the signaling mechanisms involved in
Avn-C-mediated LTP restoration. Aβ treatment inhibits LTP by activat-
ing caspase-3/GSK3β signaling and pro-inflammatory markers. Avn-C
blocks these signaling pathways and rescues LTP by inducing AMPK
phosphorylation through binding to α-1A AR. Pretreatment with either
Compound C or α-1A AR irreversible inhibitor prazosin hydrochloride
blocks the LTP-restoring effect of Avn-C
beneficial effects of Avn-C are correlated with its ability
to modulate α-1A AR, activate AMPK, and downregu-
late the factors involved in neuroinflammation.
Synaptic dysfunction is the early and major contributor to
learning and memory impairment in AD. Finding therapeutic
agents that facilitate the synaptic strengthening would be ben-
eficial for memory retrieval in AD. With that in mind, initially
we checked the effect of Avn-C on hippocampal LTP in
Tg2576 and 5XFAD mice. Both these mice overexpress mu-
tant form of human APP and shown to have early LTP impair-
ment [46–48]. LTP induction in both transgenic mice was not
altered by Avn-C treatment but the LTP maintenance was
enhanced in case of Avn-C-treated slices compared to its un-
treated control. LTP maintenance requires strengthening of
synapses and this data suggests Avn-C promotes it. Caspase-
3 reported to inhibit LTP and promotes LTD in Aβ-treated
hippocampi [7, 49, 50]. We also observed increased levels of
cleaved caspase-3 in both AD mice hippocampi and Avn-C
treatment reduced its levels which correlate with increased
LTP. Moreover, LTP analyses and immunoblot data from
2 weeks orally administered Tg2576 and 5XFAD mice
showed similar LTP enhancement and cleaved caspase-3 re-
duction levels. In addition, Avn-C oral administration im-
proved hippocampus-dependent recognition and spatial mem-
ory in 5XFAD mice.
We next investigated whether the Avn-C-induced memory
retrieval could be the results of its effect on amyloid pathology
in these mice. Thus, we examined the levels of BACE1, APP,
soluble Aβ, and phosphorylated tau using immunoblot and
IHC staining. Aβ-related proteins and phosphorylated tau
remained unchanged by Avn-C treatment in 5XFAD and
Tg2576 mice, suggesting that the protective effects are not
Aβ or tau dependent. Nonetheless, Avn-C administration
significantly reduced the inflammatory processes in these
mice. Inflammatory processes are thought to have an active
role in AD formation and progression. Multiple lines of evi-
dence showed elevated levels of pro-inflammatory markers
such as IL-1β, IL-6, TNFα, and activated microglia in AD
patients CSF and brain samples [51–54]. Aβ-induced elevated
levels of TNFα showed to impair LTP in mouse hippocampi
[55, 56]. Inhibition of dysregulated inflammatory pathways
showed to improve memory in AD mice [57, 58]. In our study,
we showed that Avn-C treatment significantly reduced the
expression of pro-inflammatory cytokines whereas it in-
creased the expression of anti-inflammatory cytokine in both
Tg2576 and 5XFAD mice hippocampi. Furthermore, our data
suggests that Avn-C exerts its effect through the activation of
AMPK. Activation of AMPK has been linked to regulate and
reduce inflammation and memory enhancement [34, 59, 60].
Orally administered resveratrol mediated AMPK activation
results in improved memory and reduced Aβ burden in
APP/PS1 mice [61, 62]. In support of this, AMPK activator
metformin treatment ameliorated cognitive impairment and
reduced Aβ burden in APP/PS1 mice [63]. Similar to these
results, our study also confirms the inhibition of AMPK with
Compound C or prazosin HCl blocked the LTP in acute hip-
pocampal slices of transgenic mouse models. Furthermore,
pull-down assay with biotinylated Avn-C-treated hippocam-
pal slice samples showed the interaction between Avn-C and
α-adrenergic receptors which was inhibited in the presence of
prazosin HCl. These results indicate that the LTP-restoring
property of Avn-C is due to activation of AMPK through α-
adrenergic receptors which results in reduced IKK, NFkB
p65, caspase-3 activation, and increased GSK3β (S9)
phosphorylation.
Noradrenergic receptors plays major role in learning and
memory process and their dysfunctions have been reported in
mild and moderate AD conditions. The α-1A ARs are G
protein-coupled receptors highly expressed in cognitive cen-
ters of the central nervous system, including the prefrontal
cortex and hippocampus [64]. These receptors are generally
stimulatory, and their role in plasticity has been documented;
binding of its ligand, norepinephrine, promotes hippocampal
LTP [39]. Furthermore, α-1A AR dysfunction is implicated in
AD. For example, polymorphisms in the α-1A AR gene are
associated with AD susceptibility [65]. In addition, long-term
stimulation of α-1A ARs increases neurogenesis and im-
proves synaptic plasticity [66]. These studies suggest that in-
creasing α-1A AR activity may delay or ameliorate AD symp-
toms. In contrast to these above observations, a small cohort
study on prazosin shown to alleviate agitation and aggressive
behavior in AD patients as a result of α-1A ARs inhibition
[67]. However, the finding in our study strongly suggests that
Avn-C may improve synaptic plasticity and behavioral defi-
cits by modifying neuroinflammation, although further studies
are required to substantiate this.

Conclusions
In conclusion, this study provides the first evidence of the
anti-AD properties of Avn-C, a natural compound that accu-
mulates and is biologically available when orally administered
to rats and humans [68, 69]. Results from our CSF analysis
also confirmed the presence of Avn-C which supports its brain
entry through blood-brain barrier membrane
(Additional file 3: Fig. S3). No apparent toxicity or adverse
effects of Avn-C were reported in these previous studies [68,
69]. Our LTP analysis data also suggests that no toxic effects
were observed even at high concentrations (Additional file 6:
Fig. S6). Presently available drugs to treat AD cause adverse
effects upon long-term dosage, which restricts their use. By
contrast, Avn-C has few or no adverse effects, making it a
potential alternative therapeutic candidate. Overall, we sug-
gest that this compound warrants development as a potential
therapeutic for AD.
Author Contributions The study was conceived by H.K. and J.J. The
experiments were designed by H.K., W.C., Y.L., and J.J. and carried out
by V.S.R., M.S., S.Y.Y., H.K.K., and S.H. The manuscript was written by
V.S.R., H.K., W.C., Y.L., and J.J. All authors read and approved the final
manuscript.
Funding Information This work was funded by the Pioneer Research
Center Program through the National Research Foundation of Korea
funded by the Ministry of Science and ICT (NRF-
2014M3C1A3053029), the Cooperative Research Program for
Agriculture Science & Technology Development (PJ010508042014,
PJ01255102, and PJ012551042019), the Rural Development
Administration, and the Republic of Korea, National Research
Foundation of Korea (NRF) grant funded by the Korea government
(NRF-2016R1A2B4008316, 2019R1A2C1004575).
Compliance with Ethical Standards
Ethics Approval and Consent to Participate Not applicable.
Consent for Publication Not applicable.
Competing Interests The authors declare that they have no conflict of
interests.
Abbreviations AD, Alzheimer’s disease; aCSF, artificial cerebrospinal
fluid; Aβ42, Amyloid β1-42; AMPK, Adenosine mono phosphate kinase;
α-1A AR, alpha 1A adrenergic receptors; CA1, cornu ammonis area 1;
CA3, cornu ammonis area 3; fEPSP, field excitatory postsynaptic poten-
tial; LTP, long-term potentiation; GSK3β, glycogen synthase kinase β;
IL, interleukin; TNFα, tumor necrosis factor-α; Tg2576 and 5XFAD,
transgenic AD mouse models
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