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7,8-Dihydroxyflavone Prevents Synaptic Loss and Memory Deficits in a Mouse Model of Alzheimer’s Disease


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Synaptic loss in the brain correlates well with disease severity in Alzheimer disease (AD). Deficits in brain-derived neurotrophic factor (BDNF)/tropomyosin-receptor-kinase B (TrkB) signaling contribute to the synaptic dysfunction of AD. We have recently identified 7,8-dihydroxyflavone (7,8-DHF) as a potent TrkB agonist that displays therapeutic efficacy toward various neurological diseases. Here we tested the effect of 7,8-DHF on synaptic function in an AD model both in vitro and in vivo. 7,8-DHF protected primary neurons from Aβ-induced toxicity, and promoted dendrite branching and synaptogenesis. Chronic oral administration of 7,8-DHF activated TrkB signaling and prevented Aβ deposition in transgenic mice that coexpress five familial Alzheimer's disease mutations (5XFAD mice). Moreover, 7,8-DHF inhibited the loss of hippocampal synapses, restored synapse number and synaptic plasticity, and prevented memory deficits. These results suggest that 7,8-DHF represents a novel oral bioactive therapeutic agent for treating AD.Neuropsychopharmacology accepted article preview online, 11 September 2013. doi:10.1038/npp.2013.243.
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7,8-Dihydroxyflavone Prevents Synaptic Loss and Memory
Deficits in a Mouse Model of Alzheimer’s Disease
Zhentao Zhang
, Xia Liu
, Jason P Schroeder
, Chi-Bun Chan
, Mingke Song
, Shan Ping Yu
David Weinshenker
and Keqiang Ye*
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA;
Department of Neurology,
Renmin Hospital of Wuhan University, Wuhan, China;
Department of Human Genetics, Emory University School of Medicine, Atlanta,
Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, USA
Synaptic loss in the brain correlates well with disease severity in Alzheimer disease (AD). Deficits in brain-derived neurotrophic factor/
tropomyosin-receptor-kinase B (TrkB) signaling contribute to the synaptic dysfunction of AD. We have recently identified 7,8-
dihydroxyflavone (7,8-DHF) as a potent TrkB agonist that displays therapeutic efficacy toward various neurological diseases. Here we
tested the effect of 7,8-DHF on synaptic function in an AD model both in vitro and in vivo. 7,8-DHF protected primary neurons from Ab-
induced toxicity and promoted dendrite branching and synaptogenesis. Chronic oral administration of 7,8-DHF activated TrkB signaling
and prevented Ab deposition in transgenic mice that coexpress five familial Alzheimer’s disease mutations (5XFAD mice). Moreover, 7,8-
DHF inhibited the loss of hippocampal synapses, restored synapse number and synaptic plasticity, and prevented memory deficits. These
results suggest that 7,8-DHF represents a novel oral bioactive therapeutic agent for treating AD.
Neuropsychopharmacology (2014) 39, 638–650; doi:10.1038/npp.2013.243; published online 2 October 2013
Keywords: 7,8-Dihydroxyflavone; Synapse; TrkB; Alzheimer’s disease; neuroprotection
The etiology and pathogenesis of Alzheimer disease (AD)
have not been fully elucidated. Neuritic plaques and
neurofibrillary tangles are the two major pathological
hallmarks of AD. However, they are only weakly correlated
with the degree of dementia in AD patients (Berg et al,
1998). It has been demonstrated that synaptic loss is the
major correlate of cognitive impairment and acts as the
pathological basis of cognitive alteration in AD brain (Sze
et al, 1997; Terry et al, 1991).
Brain-derived neurotrophic factor (BDNF) is the most
wildly distributed neurotrophin in the central nervous
system. By binding to its specific cognate receptor tropo-
myosin-receptor-kinase B (TrkB), BDNF triggers the activa-
tion of Ras/Raf/MAKP, PLCg, and PI3K/Akt signaling
cascades, which have critical roles in neuronal plasticity,
survival and neurogenesis (Diniz and Teixeira, 2011; Zuccato
and Cattaneo, 2009). It has been documented that BDNF
expression is reduced in the hippocampus, dentate gyrus,
neocortex, and in the nucleus basalis of Meynert of AD
patients (Murer et al, 2001; Narisawa-Saito et al,1996;
Phillips et al, 1991). Interestingly, neurons containing
neurofibrillary tangles do not contain BDNF immunoreac-
tivity, whereas intense BDNF-positive neurons are devoid of
tangles (Murer et al, 1999). These studies suggest that BDNF
may have a protective role against AD pathogenesis. In
agreement with this hypothesis, BDNF gene delivery reversed
synapse loss and cognitive deficits in both a rodent and a
primate model of AD (Nagahara et al,2009).However,the
outcomes of several clinical trials using recombinant BDNF
to treat amyotrophic lateral sclerosis have been disappointing
(Beck et al,2005;Ochset al, 2000), presumably due to poor
delivery and the short in vivo half-life of BDNF.
To search for a TrkB agonist with better bioavailability,
blood–brain barrier penetration, and half-life than BDNF,
we screened thousands of compounds from a chemical
library. After extensive validation, we identified 7,8-
dihydroxyflavone (7,8-DHF) as a selective small-molecular
TrkB agonist that mimics the physiological actions of
BDNF. Our pharmacokinetic experiments found that the
oral bioavailablity of 7,8-DHF is about 5%, and its half-life
is about 134 min in the plasma after oral gavage of 50 mg/kg.
Furthermore, it can penetrate the blood–brain barrier (Liu
et al, 2013). Remarkably, systemic administration of 7,8-
DHF can activate TrkB receptors in the brain as evidenced
by increases in phosphorylated TrkB in rodents (Ander o
et al, 2012; Andero et al, 2011; Choi et al, 2010 ; Jang et al ,
2010; Liu et al, 2010). These findings provide a proof-of-
concept demonstration that 7,8-DHF is a novel small
molecule for the therapeutic intervention of AD.
*Correspondence: Dr K Ye, Department of Pathology and Laboratory
Medicine, Emory University School of Medicine, 145 Whitehead Bldg.,
615 Michael St, Atlanta, GA 30322, USA, Tel: +404 712 2814, Fax:
+404 712 2979, E-mail:
These authors contributed equally to this work.
Received 14 June 2013; revised 29 August 2013; accepted 4
September 2013; accepted article preview online 11 September 2013
Neuropsychopharmacology (2014) 39, 638650
2014 American College of Neuropsychopharmacology. All rights reserved 0893-133X/14
It was recently reported that 7,8-DHF, when administered
to the 5XFAD mouse model of AD at 12–15 months of age
via daily intraperitoneal (i.p.) injection for 10 days, restored
TrkB signaling, decreased b-secretase, reduced Ab levels,
and rescued Y-maze performance (Devi and Ohno, 2012). In
the present study, we first examined the effect of 7,8-DHF on
Ab-induced neurotoxicity and synaptogenesis in vitro. Nest,
we administered 7,8-DHF orally via drinking water to the
5XFAD transgenic mouse model of AD, which contains five
familial Alzheimer’s disease mutations (Swedish, Florida,
and London human APP 695 mutations and M146L and
L286V PS1 mutations). Treatment started at 2 months of
age, before plaque deposition, and mice were assessed for
AD-like neuropathology and cognitive performance at 6
months of age. We found that 7,8-DHF protected the
primary cortical neurons and locus coeruleus (LC) neurons
from Ab-induced toxicity and promoted dendritic growth
and synaptogenesis. In 5XFAD mice, chronic oral adminis-
tration of 7,8-DHF prevented Ab deposition, the loss of
hippocampal synapses, synaptic dysfunction, and spatial
memory deficits.
Mice and Reagents
5XFAD mice on a C57BL/6J background were obtained from
the Jackson laboratory (Bar Harbor, ME) and were bred in a
pathogen-free environment in accordance with Emory
Medical School guidelines. The mice received vehicle or
7,8-DHF in their drinking water. To dissolve 7,8-DHF in
water, 1M NaOH was added drop wise to the water and
stirred at room temperature overnight. The final concentra-
tion of 7,8-DHF was 22 mg/l (pH 7.6–7.8). Water (pH 7.6–
7.8) was used as vehicle control. As the daily water intake of
C57BL/6J mice is about 7 ml/30 g body weight (Bachmanov
et al, 2002), the oral dose of 7,8-DHF is B5 mg/kg/da y. The
treatment started at 2 months of age, and mice were
subjected to Morris water maze tests at 6 months of age.
After behavior testing, the mice were killed for immuno-
blotting, imm unohistochemistry, and ELISA experiments.
Anti-TrkB antibody was purchased from Biovision (Milpi-
tas, CA). Anti-phospho-TrkB Y816 antibody was raised
against [H]-CKLQNLAKASPV-pY-LDILG-[OH] (a.a. 806–
822)(EM437 and EM438) as rabbit polyclonal antibody.
Anti-synaptotagmin, anti-MAP2, anti-Ab, and anti-tubulin
were purchased from Sigma -Aldrich (St Louis, MO). Anti-
synapsin I, anti-PSD95, anti-spinophilin, anti-Akt, anti-p-
Akt, anti-ERK, and anti-phospho-ERK1/2 antibodies were
purchased from Cell Signaling (Boston, MA). Anti-vesicular
GABA transporter (VGAT) was purchased from Thermo
Scientific (Hudson, NH). Anti-bassoon was purchased from
Stressgen (Kampenhout, Belgium). Anti-VGAT w as pur-
chased from Thermo Scientific. Anti-bassoon was pur-
chased from Stressgen. Anti-tyrosine hydroxylase (TH) was
from Calbiochem (Darmstadt, Germany). Synthetic Ab (1–
42) was purchased from rPeptide (Bogart, GA) and was
dissolved in N2 medium at 0.5 mg/ml and incubated for
4 days at 37 1C to pre-aggregate the peptide. Histostain-SP
and Ab 1–42 ELISA kits were purchased from Invitrogen
(Grand Island, NY). The In situ cell death detection kit was
purchased from Roche (Indianapolis, IN). 7,8-DHF was
purchased from TCI (Portland, OR). All chemicals not
included above were purchased from Sigma-Aldrich.
Primary Neuron Culture
Primary rat cortical neurons and LC neurons were cultured
as previously described (Chan et al, 2011). To measure the
effect of 7,8-DHF on dendrite elongation, neurons cultured
3daysin vitro (DIV 3) were exposed to 500 nM 7,8-DHF or
vehicle for 3 days, the neurons were then fixed in 4%
formaldehyde, permeabilized, and immunostained with anti-
MAP2 antibody. Pictures of the neurons were taken by
fluorescence microscopy. Dendritic length and complexity
were scored using computer software ImageJ (National
Institute of Health, USA) as described (Chan et al, 2011).
Data analysis were performed using Student’s t-test. To
assess the effect of 7,8-DHF on Ab-induced toxicity, 7,8-DHF
(500 nM) was added to the medium 20 min before Ab
treatment. Then cortical neurons and LC neurons were
exposed to 20 mMpre-aggregatedAb (1–42) and Ab (25–35),
respectively and incubated for 18 h. Neuronal apoptosis was
detected with the in situ cell death detection kit. The
apoptotic index was expressed as the percentage of TUNEL-
positive neurons out of the total number of MAP2-positive
Immunofluorescence and Immunohistochemistry
For immunofluorescence and TUNEL staining, the sections
were incubated overnight at 4 1C with anti-MAP2 antibody.
After being washed with tris-buffered saline, the sections
were incubated with Alexa Fluor 488-coupled secondary
antibodies. The sections were then incubated with TUNEL
reagent for 1 h at room temperature. After a phosphate-
buffered saline (PBS) wash, images were acquired through
an AxioCam camera on an Axiovert 200M microscope
(Zeiss). For the analysis of synaptogenesis in primary
cultured neurons, the neurons were costained with anti-
VGAT and anti-bassoon antibody. The number and size of
the synapses were analyzed with ImageJ software. Immuno-
histochemistry was performed according to the manufac-
turer’s instructions (no. 956143 and no. 956543, Invitrogen).
Briefly, tissue sections were deparaffinized and hydrated.
After antigen-retrieval in boiling 10 mM sodium citrate (pH
6.0) for 20 min, the sections were incubated with primary
antibodies (anti-trkb, anti-p-TrkB, or anti-Ab) overnight at
4 1C. The signal was developed using Histostain-SP kit. The
number of positive cells or plaques was analyzed using
ImageJ software (National Institute of Health) and color
deconvolution plugin as described previously (Josephs et al,
2008). First, color deconvolution was used to isolate AEC
stain, which represents the positive area from the hematox-
ylin stain that represents the nuclei. Image was changed into
8-bit type (gray), and then processed into binary color
image. Number of positive neurons or plaques was
calculated using the ‘analyze particles’ plugin of Imagine J.
Golgi Stain
Mice brains were fixed in 10% formalin for 24 h and then
immersed in 3% potassium bichromate for 3 days in the
dark. The solution was changed each day. Then the brains
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
were transferred into 2% silver nitrate solution and
incubated for 24 h in the dark. Vibratome sections were
cut at 60 mm, air dried for 10 min, dehydrated through 95
and 100% ethanol, cleared in xylene, and coverslipped. For
measurement of spine density, only spines that emerged
perpendicular to the dendritic shaft were counted.
Western Blot Analysis
The mice brain tissue was lysed in lysis buffer (50 mM Tris,
pH 7.4, 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100,
1.5 mM Na
, 50 mM NaF, 10 mM sodium pyropho-
sphate, 10 mM sodium b-glycerophosphate, supplemented
with protease inhibitors cocktail) and centrifuged for
15 min at 16 000 g. The supernatant was boiled in SDS
loading buffer. After SDS-PAGE, the samples were trans-
ferred to a nitrocellulose membrane. Western blotting
analysis was performed with a variety of antibodies.
Electron Microscopy
Synaptic density was determined by electron microscopy as
described previously (Hongpaisan et al, 2011). After deep
anesthesia, mice were perfused transcardially with 2%
glutaraldehyde and 3% paraformaldehyde in PBS. Hippo-
campal slices were postfixed in cold 1% OsO
for 1 h. Samples
were prepared and examined using standard procedures.
Ultrathin sections (90 nm) were stained with uranyl acetate
and lead acetate and viewed at 100 kV in a JEOL 200CX
electron microscope. Synapses were identified by the
presence of synaptic vesicles and postsynaptic densities.
Electrophysiological Analysis
Electrophysiological analysis was carried out as previously
described (Hong et al, 2012). Briefly, vehicle- and 7,8-DHF-
treated 5XFAD mice were anaesthetized with isoflurane,
decapitated, and the hippocampi were cut into 400-mm thick
transverse slices with a vibratome. After incubation at room
temperature in a-CSF for 60–90 min, slices were placed in a
recording chamber on the stage of an upright microscope
(Olympus CX-31) and perfused at a rate of 3 ml per min
with a-CSF (containing 1 mM MgCl
) at 23–24 1C. A 0.1 MO
tungsten monopolar electrode was used to stimulate the
Schaffer collaterals. The field excitatory postsynaptic poten-
tials (fEPSPs) were recorded in CA1 stratum radiatum by
a glass microelectrode filled with a-CSF with resistance of
3–4 MO. The stimulation output (Master-8; AMPI, Jerusa-
lem) was controlled by the trigger function of an EPC9
amplifier (HEKA Elektronik, Lambrecht, Ger many). fEPSPs
were recorded under current-clamp mode. Data were
filtered at 3 kHz and digitized at sampling rates of 20 kHz
using Pulse software (HEKA Elektronik). The stimulus
intensity (0.1 ms duration, 2–4 V) was set to evoke 40% of
the maximum fEPSP and the test pulse was applied at a rate
of 0.033 Hz. Field potential input-output curves were
constructed by measuring fEPSP slope responding to the
stimulus intensity increased from 1 to 7 V, with an 0.5 V
increment. LTP of fEPSPs was induced by 3 theta-burst-
stimulation (TBS), it is 4 pulses at 100 Hz, repeated 3 times
with a 200-ms interval). Paired-pulse facilitati on (PPF) was
examined by applying pairs of pulses, which were separated
by 20–500 ms intervals. The magnitudes of LTP are expressed
as the mean percentage of baseline fEPSP initial slope.
Ab Plaque Staining
Amyloid plaques were stained with Thioflavin-S. The
deparaffinized and hydrated sections were incubated in
0.25% potassium permanganate solution for 20 min, rinsed
in distilled water, and incubated in bleaching solution
containing 2% oxalic acid and 1% potassium metabisulfite
for 2 min. After rinsing in distilled water, the sections were
transferred to blocking solution containing 1% sodium
hydroxide and 0.9% hydrogen peroxide for 20 min. The
sections were incubated for 5 s in 0.25% acidic acid, then
washed in distilled water and stained for 5 min with
0.0125% Thioflavin-S in 50% ethanol. The sections were
washed with 50% ethanol and placed in distilled water. Then
the sections were covered with a glass cover using mounting
solution. Quantitative assessment of plaque areas was done
using ImageJ software as described previously (Heneka
et al, 2013). Briefly, the images were normalized and an
automatic thresholding on the basis of the entropy of the
histogram was used to identify the plaques. The pictures
were converted to a binary, and then the plaque number
and the plaque area were calculated using the ‘anal yze
particles’ plugin of Imagine J.
The mice brains were homogenized in 8X mass of 5 M
guanidine HCl/50 mM Tris HCl (pH 8.0) and incubated at
room temperature for 3 h. Then the samples were diluted
with cold reaction buffer (PBS with 5% BSA and 0.03%
Tween-20, supplemented with protease inhibitor cocktail),
and centrifuged at 16 000 g for 20 min at 4 1C. The super-
natant was analysed with a human Ab42 ELISA kit
according to the manufacturer’s instructions (KHB3441,
Invitrogen). The Ab42 concentrations were determined by
comparison with the standard curve.
Morris Water Maze
Female wild-type and 5XFAD mice maintained on standard
drinking water or 7,8-DHF were trained in a round, water-
filled tub (52 inch diameter) in an environment rich with
extra maze cues. An invisible escape platform was located in
a fixed spatial location 1 cm below the water surface
independent of a subjects start position on a particular
trial. In this manner, subjects needed to utilize extra maze
cues to determine the platform’s location. At the beginning
of each trial, the mouse was placed in the water maze with
their paws touching the wall from one of four different
starting positions (N, S, E, and W). Each subject was given
four trials/day for 5 consecutive days with a 15-min
intertrial interval. The maximum trial length was 60 s and
if subjects did not reach the platform in the allotted time,
they were manually guided to it. Upon reaching the invisible
escape platform, subjects were left on it for an additional 5 s
to allow for survey of the spatial cues in the environment to
guide future navigation to the platform. After each trial,
subjects were dried and kept in a dry plastic holding cage
filled with paper towels to allow the subjects to dry off. The
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
temperature of the water was monitored every hour so that
the mice were tested in water that was between 22 and 25 1C.
Following the 5 days of task acquisition, a probe trial was
presented during which time the platform was removed and
the percentag e of time spent in the quadrant that previously
contained the escape platform during task acquisition was
measured over 60 s. All trials were analysed for latency,
swim path length, and swim speed by means of MazeScan
(Clever Sys).
Statistical Analysis
All of the data were presented as mean
SEM. Statistical
analysis were performed using either Student’s t-test (two-
group comparison) or one-way ANOVA followed by the
Dunnett’s post hoc multiple comparison test (mor e than two
groups). The level of significance was set for P-valueo0.05.
7,8-DHF Protects Primary Cortical Neurons and LC
Neurons from Ab-Induced Toxicity in a TrkB-Dependent
We previously reported that 7,8-DHF displays protective
effect at 500 nM, and it induces TrkB tyrosine phosphoryla-
tion in hippocampal neurons (Jang et al, 2010; Liu et al,
2013). To examine whether 7,8-DHF has protective effects
on neurons against Ab-induced toxicity, we prepared rat
primary cortical neurons and challenged them with pre-
aggregated Ab (1–42). The slides were immunostained with
the neuronal marker MAP2 and then stained with TUNEL
in situ cell death detection Kit. Ab treatment provoked
neuronal apoptosis as indic ated by TUNEL assay. Neuronal
apoptosis induced by Ab was substantially blocked by
500 nM 7,8-DHF (Figures 1a and c). Some of the positive
TUNEL signals were not overlapped with MAP2 (white
arrow), indicating that other cell types but not neurons were
dead. It has been demonstrated that AD patients present
with an early and prominent loss of LC neurons
(Chalermpalanupap et al, 2013; Matthews et al, 2002;
Weinshenker, 2008). Accordingly, we also tested the effect
of 7,8-DHF on Ab-induced toxicity in LC neurons. 7,8-DHF
significantly attenuated neuronal death induced by pre-
aggregated Ab. To determine whether the protective effect
of 7,8-DHF is dependent on TrkB activity, we pretreated the
neurons with K252, a Trk receptor inhibitor 30 min before
7,8-DHF introduction. K252 pretreatment completely
blocked the protective effect of 7,8-DHF (Figures 1b and
d), demonstrating that the protective effect of 7,8-DHF is
dependent on TrkB activity.
7,8-DHF Promotes Dendrite Branching and Synaptic
Formation in vitro
BDNF is req uired for normal dendritic morphology and
synapse formation and maintenance (Gomes et al, 2006;
Hiester et al, 2013). In order to test whether 7,8-DHF affects
dendrite elongation, we assessed the dendritic length in the
presence or absence of 7,8-DHF. We found that exposure to
500 nM 7,8-DHF for 3 days significantly increased the total
dendritic length of primary cultured neurons (Figures 2a
and b). We also tested the number of crossings between
dendrites and calculated the area under the curve (AUC).
The number of dendritic crossings was significantly
increased by 7,8-DHF. These results indicate that 7,8-DHF
promotes dendritic branching (Figure 2c).
To further address the effect of 7,8-DHF on synaptic
formation and maintenance in vitro, we stained vehicle- or
7,8-DHF-treated neurons with the presynapti c markers
VGAT and bassoon. The density and structure of pre-
synaptic structures were examined using immunofluores-
cent confocal microscopy. 7,8-DHF treatment increased the
number of presynaptic structures expressing VGAT and
bassoon. 7,8-DHF also increased synapse size (Figures
3d–f). These results suggest that 7,8-DHF promotes
dendritic arborization and facilitates the formation and
maintenance of synapses in vitro.
Chronic Oral Administration of 7,8-DHF Activates TrkB
and Downstream Signaling Pathways in 5XFAD Mice
To explore whether oral administration of 7,8-DHF can
activate TrkB in mouse brain, we supplied 7,8-DHF or
vehicle in the drinking water of non-transgenic beginning at
2 months of age. After 4 months of drug treatment, we
monitored TrkB activation in the brain by immunohisto-
chemistry with anti-phosphorylated TrkB (p-TrkB) anti-
body. Quantitative analysis revealed that p-TrkB-positive
neurons in 5XFAD mice appeared to be fewer compared with
non-transgenic control, but the decrease was not statistically
significant. However, 7,8-DHF treatment significantly ele-
vated p-TrkB but not total TrkB levels in 5XFAD mice
(Figures 3a and b). This result was confirmed by immuno-
blotting of the brain lysates. BDNF treatment elicits robust
TrkB tyrosine phosphorylation and activates several down-
stream signaling pathways, such as PI3K/AKT and Ras/Raf/
MAPK pathways. As expected, the TrkB receptor was more
prominently phosphorylated in 5XFAD mice treated with
7,8-DHF than vehicle control, as were the downstream AKT
and ERK/MAPK pathways (Figure 3c). Given the fact that
7,8-DHF can pass the blood–brain barrier (Liu et al, 2013),
these results suggest that chronic oral administration of 7,8-
DHF directly activates TrkB receptor and its downstream
signaling pathways in the brain.
Chronic Oral Administration of 7,8-DHF Prevents
Synaptic Loss in 5XFAD Mice
Synaptic loss is believed to be the basis of cognitive
impairment in the early phase of AD (Scheff et al, 2011). In
5XFAD mice, significant synaptic loss and behavior deficits
were detected at 5 months of age, in the absence of
detectable neuronal loss (Hongpaisan et al, 2011). We first
assessed the density of dendritic spines along individual
dendrites of pyramidal neurons by Golgi stain. The density
of dendritic spines was markedly decreased in the 5XFAD
mouse model compared with the non-transgenic group, and
this deficit was markedly rescued by 7,8-DHF treatment
(Figures 4a and b). As each dendritic spine can form more
than one synapse, we directly quantified the density of
synapses in the CA1 area of 5XFAD mice brain by electron
microscopy. 5XFAD mice showed a significant reduction
in synaptic density compared with non-transgenic control
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
mice. 7,8-DHF treatment notably reversed the loss of
synaptic density (Figures 4c and d). We further confirmed
these findings by immunoblotting using presynaptic
markers (synaptotagmin and synapsin I) and postsynaptic
markers (PSD95 and spinophilin). 5XFAD mice showed a
significant decrease in these synaptic markers, indicating
synaptic degeneration. 7,8-DHF treatment reversed the
decrease of synaptic markers (Figure 4e). These results
suggest that activation of TrkB receptors by 7,8-DHF
inhibits the loss of synapse in 5XFAD mice.
Chronic Oral Administration of 7,8-DHF Restores
Synaptic Plasticity in 5XFAD Mice
Long-term potentiation (LTP) is a measu re of synaptic
plasticity that underlies learning and memory. 5XFAD mice
show significantly impaired LTP at the Schaffer collateral-
CA1 pathways compared with non-transgenic mice (Kimura
and Ohno, 2009). We ex amined whether chronic oral 7,8-
DHF treatment can rescue LTP deficit observed in
hippocampal slices from 5-month-old 5XFAD mice. As
shown in Figure 5a, LTP magnitude was significantly
decreased compared with the non-transgenic control mice,
and the decreased LTP magnitude in 5XFAD mice was
significantly reversed by 7,8-DHF treatment, suggesting a
definite enhancement in synaptic plasticity by 7,8-DHF. We
also tested PPF, which represents a short-term form of
synaptic plasticity reflecting presynaptic function. Consis-
tent with the previous report that PPF was not affected in
5XFAD mice (Kimura and Ohno, 2009), we found that the
ratio of paired pulse was similar when comparing non-
transgenic control, vehicle- and 7,8-DHF-treated 5XFAD
Figure 1 7,8-dihydroxyflavone (7,8-DHF) prevents Ab-induced neurotoxicity in cultured cortical neurons and locus coeruleus (LC) neurons. (a) 7,8-DHF
protected cortical neurons form Ab toxicity. Cultured cortical neurons (DIV 12) were exposed to pre-aggregated Ab (1–42, 20 mM) for 18 h in the presence
or absence of 7,8-DHF (500 nM). Neurons were immunostained with neuronal marker MAP2. Neuronal apoptosis was detected by TUNEL staining. The
neurons in apoptosis were indicated by white arrows. (b) 7,8-DHF protects LC neurons from Ab toxicity. LC neurons were exposed to pre-aggregated Ab
(25–35, 20 mM) for 18 h in the presence or absence of 7,8-DHF (500 nM) and the Trk receptor inhibitor K252 (100 nM), and stained with tyrosine
hydroxylase (TH) (red), DAPI (blue) and TUNEL (green). The percentage of apoptotic cells was determined by TUNEL staining. (c) Quantification of
TUNEL-positive cells show that 7,8-DHF decreased the apoptotic rate of cortical neurons induced by Ab. Data represent the mean
SEM from three
independent experiments. *Po0.01. (d) Quantification of TUNEL-positive neurons shows that 7,8-DHF attenuated Ab-induced apoptosis in LC neurons.
The protective effect of 7,8-DHF was abolished by K252. Data represent the mean
SEM from three independent experiments. *Po0.01.
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
mice (Figure 5b). Then we tested the basal synaptic
transmission at CA1 synapses by measuring the input/
output (I/O) curves. We found that the basal synaptic
transmission was impaired in 5XFAD mice, and the I/O
curve was not affected by 7,8-DHF treatment, indicating
that 7,8-DHF did not rescue the basal synaptic transmission
in 5XFAD mice (Figures 5c and d).
Chronic Oral Administration of 7,8-DHF Alleviates Ab
Deposition But not The Concentration of Total Ab
We further investigated the effect of 7,8-DHF treatment on
senile plaque formation by Thiofla vin-S staining. 5XFAD
mice show significant plaque deposition at 6 months of age.
Strikingly, the number of plaques in the hippocampus was
significantly decreased in 7,8-DHF-treated mice as com-
pared with vehicle control (Figures 6a and b). We further
tested the deposition of Ab by immunohistochemistry with
anti-Ab antibody. Ab deposition was lower in 7,8-DHF-
treated group when compared with mice given standard
drinking water (Figures 6c and d). We also measured the
concentrations of total Ab42 by ELISA. Interestingly, total
Ab42 concentration in 5XFAD mice was not affected by
7,8-DHF treatment (Figure 6e). These results suggest that
chronic oral 7,8-DHF may prevent Ab deposition and
plaque formation but not Ab production.
Chronic Oral Administration of 7,8-DHF Rescues
Memory Deficits in 5XFAD Mice
Hippocampal-dependent spatial memory of 5XFAD mice at
6 months of age was tested with the invisible platform task
in the Morris water maze test. The average latency
(Figure 7a), swim path length (Figure 7b), and swim speed
(Figure 7c) for each of the 5 acquisition days were calculated
and plotted. A two-way mixed analysis of variance
(ANOVA) (Group Training Day (repeated measure)) on
latency revealed a main effect of training day (F (4,
156) ¼ 15.29, Po0.05) and group (F (3, 39) ¼ 3.88,
Po0.05) but not interaction (F (12, 156) ¼ 0.4423, ns). A
mixed two-way ANOVA on swim path distance revealed a
significant main effect of training day (F (4, 156 ¼ 14.81,
Po0.05)) but no effect of group (F (3, 39) ¼ 0.84, ns) or
interaction (F (12, 156) ¼ 1.16, ns). Separate one-way
repeated measures ANOVAs on swim path distance revealed
a significant effect of training day for water-treated wild-
type (F (4, 40) ¼ 2.77, Po0.05), 7,8-DHF-treated wild-type
(F (4, 28) ¼ 10.71, Po0.05), and 7,8-DHF-treated 5XFAD
Figure 2 7,8-dihydroxyflavone (7,8-DHF) promotes synaptogenesis in primary cultured neurons. (a) Representative image of primary cortical neurons.
The neurons were cultured in the presence or absence of 7,8-DHF (500 nM) for 3 days and immunostained with antibody to neuronal marker MAP2.
(b) Quantification of total dendritic length. 7,8-DHF promoted dendritic elongation in primary neurons. (c) The number of crossings and area under the
curve (AUC) following 7,8-DHF treatment.(d) The presynaptic structure of cultured neurons. Vehicle- or 7,8-DHF-treated neurons were double stained
with the presynaptic markers VGAT (green) and bassoon (red). The number of synapses (e) and synapse size (f) was quantified. 7,8-DHF significantly
promoted the number and size of neurons. Data represent the mean
SEM from three independent experiments. *Po0.01.
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
(F (4, 52) ¼ 6.48, Po0.05) mice, indicating significant
learning over the 5 days of water maze training. Conversely,
water-treated 5XFAD mice (F (4, 36) ¼ 0.58, ns) did not
demonstrate this improvement. These results indicate that
5XFAD mice had impaired acquisition of the spatial
learning task that was corrected by 7,8-DHF. A two-way
mixed ANOVA on swim speed revealed a significant
interaction between group and training day (F (12,
156) ¼ 2.387, Po0.05), and post hoc analysis revealed some
small but inconsistent differences on various days. Analysis
of the main effects of group on swim speed (F (3,
39) ¼ 3.412) revealed that, collapsed over the 5 training
days, the only consistent effect of group on swim speed was
that 7,8-DHF-treated wild-type mice swam significantly
slower than 7,8-DHF-treated 5XFAD mice. Thus, it is highly
unlikely that genotype or treatment effects on swim speed
contributed to overall task performance, particularly as
swim path distance is independent of swim speed.
Assessment of memory recall for the platform location on
the probe trial revealed impaired memory in water-treated
5XFAD mice and its rescue by 7,8-DHF (Figure 7d). A two-
way ANOVA (Genotype Treatment) performed on the
percentage of time spent in the target quadrant during the
60 s probe trial indicated a significant interaction (F (1,
41) ¼ 2.293, Po0.05). Sidak’s multiple comparisons re-
vealed that, when compared with water-treated wild-type
mice, water-treated 5XFAD mice spent a significantly lower
percentage of their time in the quadrant that form erly
contained the hidden platform. Comparison of the percen-
tage of time spent in the target quadrant between water- and
7,8-DHF-treated 5XFAD mice revealed a significant differ-
ence between these group s (t (1,22) ¼ 2.293, Po0.05),
demonstrating rescue of spatial memo ry recall by 7,8-DHF.
Our results in the present study demonstrate that 7,8-DHF
mimics BDNF and displays neurotrophic actions by
protecting cortical neurons and LC neurons from
Figure 3 7,8-dihydroxyflavone (7,8-DHF) elicits tropomyosin-receptor-kinase B (TrkB) and downstream signaling activation in 5XFAD mice.
(a) Immunohistochemistry staining for TrkB and p-TrkB in 5XFAD brain sections. Two months old 5XFAD mice were treated with 7,8-DHF (5 mg/kg/day)
or vehicle consecutively for 4 months. The phosphorylation of TrkB in dentate gyrus was detected by immunohistochemistry with anti-TrkB and anti-p-TrkB
816 antibody. Arrows indicate the p-TRKB-positive cells. Scale bar, 50 mm. (b) Quantification of p-TrkB-positive neurons in the dentate gyrus. Note that
7,8-DHF treatment elicited the phosphorylation of TrkB in 5XFAD mice. Data are shown as mean
SEM (n ¼ n ¼ 3 mice per group). *Po0.01.
(c) Immunoblotting analysis of the phophorylation of TrkB and its downstream signaling pathways Akt and ERK/MAPK. The level of p-TrkB, p-AKT and
p-ERK/MAPK was increased by 7,8-DHF treatment, indicating that 7,8-DHF elicits TrkB and its downstream signaling pathways. n ¼ 3 mice per group.
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
Ab-induced toxicity, promotes dendritic arborization, and
synaptogenesis in vitro. In the 5XFAD mouse model,
chronic oral administration of 7,8-DHF activated TrkB
signaling pathways in the brain, attenuated synaptic loss,
reversed synaptic plasticity, Ab deposition, and rescued
spatial memory deficits. These results suggest that 7,8-DHF
simulates the physiological actions of BDNF and prevents
the synaptic dysfuncti on and cognitive deficits in a rodent
model of AD.
Impairment of the BDNF-TrkB pathway has long been
believed to have an essential role in AD pathogenesis.
Accumulating evidence supports that BDNF provides a
novel therapeutic strategy for AD treatment (Arancibia
et al, 2008; Nagahara et al, 2009). Nevertheless, BDNF is
a 25 KDa protein, and as such, possesses the intrinsic
drawbacks of any polypeptide as a pharma cological agent,
including in vivo instability and poor pharmacokinetics.
To circumvent these issues, we screened thousands of
compounds and successfully identified a small molecule
7,8-DHF, which selectively provokes TrkB but not TrkA
activation in mouse brain upon i.p. or oral administration
(Andero et al, 2012; Andero et al , 2011; Jang et al, 2010;
Liu et al, 2010). We also found many flavone derivatives,
such as chrysin (5,7-DHF) fails to active TrkB receptor,
indicating that the 8-position hydroxy group is essential for
flavone derivatives to bind TrkB receptor (Jang et al , 2010).
Figure 4 7,8-dihydroxyflavone (7,8-DHF) prevents the synaptic loss in hippocampal CA1 area of 5XFAD mice. (a) Golgi staining reveals the dendritic spines
from apical dendritic layer of the CA1 region. Scale bar, 5 mm. (b) Quantitative analysis of the spine density. The decreased spine density in 5XFAD mice was
reversed by 7,8-DHF. n ¼ 6 in each group, *Po0.01. (c) Quantitative analysis of the synaptic density in non-transgenic, vehicle- and 7,8-DHF-treated 5XFAD
mice. 5XFAD mice show decreased synaptic density, which was reversed by 7,8-DHF. Data are shown as mean
SEM (n ¼ 3 mice per group). *Po0.01.
(d) Representative electron microscopy of the synaptic structures. Arrows indicate the synapses. (e) Immunoblot analysis of synaptic markers in brain
homogenates from mice treated with vehicle or 7,8-DHF. The expression of presynaptic markers (synaptotagmin and synapsin I) and post-synaptic markers
(PSD95 and spinophilin) were reduced in 5XFAD mice, indicating synaptic degeneration. 7,8-DHF reversed the decrease of synaptic markers.
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
Figure 5 7,8-dihydroxyflavone (7,8-DHF) restores synaptic plasticity in 5XFAD mice. (a) Long-term potentiation (LTP) of field excitatory postsynaptic
potentials (fEPSPs) was induced by 3xTBS (theta-burst-stimulation) (four pulses at 100 Hz, repeated three times with a 200-ms interval). Shown traces are
representative fEPSPs recorded at the time point 1 and 2 (vehicle treated), 3 and 4 (7,8-DHF-treated mouse). The magnitude of LTP in 5XFAD mice is
significantly lower than in non-transgenic control mice, and 7,8-DHF treatment reversed the LTP impairment. n ¼ 6 in each group. Data are presented as
SEM. *Po0.05 vs vehicle-treated mice. (b) The paired-pulse facilitation (PPF) of non-transgenic, vehicle-treated 5XFAD, and 7,8-DHF-treated
5XFAD mice. The ratio of paired pulse is similar between two groups. The inter-pulse interval for paired pulse is 20, 50, 100, 200, and 500 ms. n ¼ 6 in each
group. (c) Synaptic transmission assessed by input/output (I/O) relation between stimuli intensity and fEPSP slope. I/O curves obtained in hippocampal slices
prepared from non-transgenic, vehicle-, and 7,8-DHF-treated 5XFAD mice. (d) Averaged slope of I/O curves is significantly greater in non-transgenic mice
(Po0.05, vs vehicle- or 7,8-DHF-treated 5XFAD mice). There is no difference between vehicle- and 7,8-DHF-treated 5XFAD mice. n ¼ 6 in each group.
Data are presented as mean
SEM and analyzed with two-way ANOVA.
Figure 6 7,8-dihydroxyflavone (7,8-DHF) alleviates Ab deposition but not the concentration of total Ab. (a) Thioflavin-S staining of amyloid plaques in
the hippocampus of 5XFAD mouse brain sections. Scale bar, 100 mm. (b and c) Quantitative analysis of amyloid plaques. The density of plaques (b) and the
percentage of area occupied by Ab deposits (c) in 5XFAD mouse brain were decreased by 7,8-DHF. n ¼ 3 mice per group, three sections per mice were
analyzed. *Po0.01. (d) Immunohistochemistry of Ab deposits in 5XFAD mice. Scale bar, 100 mm. (e) Quantitative analysis of amyloid plaques. Amyloid
deposition in 5XFAD mice was significantly decreased by 7,8-DHF. n ¼ 3 mice per group, three sections per mice were analyzed. *Po0.01. (f) Ab42 ELISA
in vehicle- and 7,8-DHF-treated 5XFAD mice. n ¼ 4 in vehicle group, n ¼ 9 in 7,8-DHF group. 7,8-DHF did not change the concentration of total Ab42.
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
Here we show that 7,8-DHF promotes both cortical and LC
neuron survival in the face of Ab-induced neurotoxicity. LC
neurons are one of the earliest populations of neurons
affected in AD and Parkinson disease, probably because that
they are more vulnerable than other neuronal populations.
Nevertheless, our in vitro result suggests that 7,8-DHF may
be used to protect these neurons in the early phase of AD.
5XFAD mice have been shown to develop cerebral
amyloid plaques at 2 months of age and show memory
impairment at 4–5 months of age (Oakley et al, 2006). It has
also been shown that the level of mature BDNF is
dramatically reduced in 5XFAD mice, beginning at 3
months of age (Devi and Ohno, 2012). Given the key roles
that BDNF-TrkB signaling has in learning and memory, we
propose that our TrkB agonist 7,8-DHF may protect
memory decline in 5XFAD mice. Here we show that TrkB
receptors in the dentate gyrus were activated by 7,8-DHF
treatment. The activation of TrkB downstream Akt and
MAPK pathways were coupled to TrkB phosphorylation.
Therefore, chronic oral administration of 7,8-DHF activates
BDNF-TrkB signaling in the brain of 5XFAD mice. Finally,
our results are consistent with the previous report that
systematic administration of 7,8-DHF triggers TrkB activa-
tion in a transgenic mouse model of AD and in cognitively
impaired aged rats by activating the TrkB signaling pathway
(Devi and Ohno, 2012; Zeng et al, 2012). Interestingly, we
noticed that 5XFAD mice did not show significant impair -
ment of basal TrkB activity. Presumably, the decreased
BDNF level is compensated by TrkB receptor hypersensi-
tivity. However, we observed that 7,8-DHF triggered TrkB
activation and exerted the protective effect. It is worth
noting that in our experiment the increase of p-TrkB
signaling is not as robust as in the Devi and Ohno’s study.
This may be due to the different basal TrkB activity. They
used 12- to 15-month-old mice, which demonstrate
significant impairment of TrkB signaling. We previously
reported that 7,8-DHF binds to TrkB and quickly induces
TrkB dimerization, phosphorylation, and the activation of
downstream signaling pathways (Andero et al, 2011; Jang
et al, 2010). Many different labs have independently
reported that 7,8-DHF activates the TrkB signaling pathway
in cortical neurons, motor neurons, retinal ganglion cells,
dopaminergic neurons, and so on (Apawu et al, 2013; Gupta
et al, 2013; Mantilla and Ermilov, 2012). All of these data
suggest that 7,8-DHF exert its neurotrophic activity through
TrkB. To confirm a small molecule indeed acts as a receptor
agonist, the best evidence might be co-crystallization.
However, the crystal structure of TrkB extracellular domain
(ECD) has not been full y solved yet, we had not yet directly
observed the co-crystal structure of the 7,8-DHF and TrkB
Synaptic dysfunction is a major pathophysiological hall-
mark in AD and other neurodegenerative diseases. Sub-
stantial evidence indicates that there is a decrease in the
number of synapses in AD patients and in patients with
amnestic mild cognitive impairment (aMCI), a prodromal
stage of AD (Scheff et al, 2011; Selkoe, 2002). Hence,
synaptic dysfunction is an early event in AD pathogenesis.
Furthermore, synaptic loss appears to be the best pathologic
correlate of dementia in AD (Sze et al, 1997; Terry et al,
1991). It has been suggested that ‘synaptoprotective’ therapy
will probably be more clinical relevance than neuroprotec-
tive therapy (Coleman et al, 2004). BDNF/TrkB signaling
has a direct role in the formation and maintenance of
Figure 7 Effects of 7,8-dihydroxyflavone (7,8-DHF) on spatial learning and memory of wild-type (WT) and 5XFAD mice. Female WT and 5XFAD mice
(n ¼ 10–14/group) given standard drinking water or 7,8-DHF dissolved in their drinking water were trained in the water maze over 5 days. Shown are
SEM latency to mount the escape platform (a), swim path length (b), and swim speed (c). A probe trial was performed on day 6 during which the
platform was removed. Shown is the mean
SEM percentage of time spent in the target quadrant (d). *Po0.05 compared with vehicle treated wild-type
Po0.05 compared with vehicle treated 5XFAD mice.
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
synapses (Gomes et al, 2006; Hiester et al, 2013). Like
BDNF, 7,8-DHF can significantly promote dendritic elonga-
tion and arborization in primary cultured cortical neurons
(Figure 2). Dendrites constitute 490% of the neuronal
surface available for synaptic contact (Coleman et al, 2004).
We also show that 7,8-DHF promotes synap togenesis.
Conceivably, our in vitro results indicate that 7,8-DHF
may provide protective effects on synapses in AD. Activa-
tion of TrkB is required for multiple aspects of neur onal
functions, including neuronal survival, morphological
change of neurons, and synaptic plasticity (Bekinschtein
et al, 2008; Diniz and Teixeira, 2011; Lu et al, 2013; Zuccato
and Cattaneo, 2009). TrkB signaling promotes the formation
of dendritic spines (Zeng et al, 2011). We observed a
decrease in dendritic spine density in the hippocampus of
5XFAD mice, and that TrkB agonist 7,8-DHF increased the
spine density in apical dendrites of CA1 neurons of
hippocampus. In agreement with this observation, 7,8-
DHF also exerted a beneficial effect on the number of
synapses in the CA1 area of 5XFAD mice. Furthermore, the
expression of synaptic markers was also increased by 7,8-
DHF treatment, indicating that 7,8-DHF possesses the
profound protective effects on synapses in vivo.
Several transgenic mouse models of AD show age-
dependent deficits in hippocampal LTP (Chapman et al,
1999; Oddo et al, 2003), which correlate with the impair-
ment in hippocampal-dependent memory. LTP is regarded
as cellular mechanism for learning and memory. In the
adult brain, the main functions of BDNF are to enhance
synaptic transmission, facilitate synaptic plasticity, and
promote synaptic growth. BDNF is also critical for LTP (Lu
et al, 2013). It has been reported that BDNF promotes
persistence of long-term memory storage through activation
of ERK signaling (Bekinschtein et al, 2008). Like BDNF, 7,8-
DHF can also promote the activation of ERK, a likely
mechanism by which 7,8-DHF rescues LTP impairments in
5XFAD mice. In the water maze test, 7,8-DHF improved
not only the acquisition of the spatial lear ning task but
also the spatial memory recall. This is consistent
with previous reports which demonstrated that 7,8-DHF
improves spatial memory in APP transgenic mice(Devi and
Ohno, 2012) and cognitively impaired aged rats (Zeng et al,
2012). We previously found that the brain 7,8-DHF
peaked at 10 min with a concentration of 50 ng/g of the
brain (about 200 nM) after oral administration of 50 mg/kg
dosage (Liu et al, 2013). Here the mice received
an approximate dose of 5 mg/kg/day. We estimate that
the brain concentration in the present study is lower than
the in vitro beneficial concentration of 500 nM. These
results suggest that chronic exposure of the brain to lower
concentration of 7,8-DHF is sufficient to exert the protective
effect in vivo.
Polymerization of Ab is believed to have a key role in the
pathogenesis of AD. We tested whether 7,8-DHF interferes
with Ab aggregation. We found that 7,8-DHF reduced the
formation of plaques as demonstrated by Thioflavin-S
staining and Ab immunohistochemistry. However, 7,8-
DHF treatment did not affect total cerebral Ab42 concen-
trations. Considering that numerous flavonoids exhibit
inhibitory activity against Ab42 aggregation (Sato et al,
2013). We cannot exclude the possibility that 7,8-DHF may
also directly inhibit the aggregation of Ab as well. Recently,
it was reported that 10 days of i.p. 7,8-DHF administration
decreased cerebral Ab concentration and BACE1 expression
in 5XFAD mice at 12–15 months old (Devi and Ohno, 2012).
One of the possible reasons for this discrepancy is the
different route of drug administration. Our oral adminis-
tration pharmacokinetics data show that 7,8-DHF’s oral
bioavailability is around 5% and i.p. injection may display
much higher levels of bioavailability (Liu et al
, 2013). This
difference might explain why p-TrkB signals in 7,8-DHF i.p.
injected mouse brains are stronger than what we have
shown in the brain lysates when 7,8-DHF was orally
administrated. Moreover, 10 days of i.p. treatment may
lead to higher drug levels, which may then suppress BACE1
expression, resulting in blockade of Ab production,
although 7,8-DHF does not inhibit BACE1 activity
directly. On the other hand, the age of the mice may also
contribute to this discrepancy. The 12–15 months old
5XFAD mice in the Devi and Ohno’s study represent an
advanced age of AD, whereas in our study the 5XFAD mice
begun to receive 7,8-DHF at 2 months of age, which
represent an earlier stage of disease progression. The
expression of BACE1 in the brains of older 5XFAD mice
is much higher than that in younger mice, which may make
it easier to demonstrate the inhibitory effect of drugs
(Zhang et al, 2009). Together, the difference in mice age
and different routes of drug administration may result in
different drug exposure in the brain, contributing to the
different effects of 7,8-DHF on BACE1 expression in the two
studies. Except for these differences, the present study
differs from the Devi and Ohno’s work in several
ways. First, we focused on the effect of 7,8-DHF on synaptic
dysfunction, because synaptic loss is the physical basis of
cognitive alteration in AD. Demonstration of the ‘synapto-
protective’ effect of 7,8-DHF will provide more clinically
relevant information. In addition, we measured the effect of
7,8-DHF using the water maze test, because it is the most
widely used test to measure hippocampal-dependent
spatial-based learning and memory, which is early and
prominent memory loss in AD patients, whereas the Devi
and Ohno’s study used Y-maze to test the general cognitive
In summary, this study demonstrates that chronic oral
administration of 7,8-DHF exerts therapeutic effect in
5XFAD mice. This effect can largely be attributed to the
protective effect of 7,8-DHF on synapses. Our study
identifies 7,8-DHF as a novel ‘synaptoprotective’ strateg y
for the treatment of AD and other neurodegenerative
The authors declare no conflict of interest.
This work was supported by a grant from the National
Institute of Health (RO1, DC010204) to K Ye, the
NIH/NIAP50 ADRC center grant to both K Ye and D
Weinshenker, and a grant from the National Natural
Science Foundation of China (No. 81100958) to Z Zhang.
7,8-DHF protects synapses in Alzheimer’s disease
Z Zhang et al
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... A study using both 3-month-old 5xFAD mice and WT littermates found significant temporal changes in gut microbiota, including decreased abundance of Firmicutes and increased Bacteroidetes in 5xFAD mice [43] . Compared with the WT mice, the 5xFAD mice also showed increased Helicobacter, Prevotella, and Sutterella. ...
... Compared with the WT mice, the 5xFAD mice also showed increased Helicobacter, Prevotella, and Sutterella. They reduced the microbial diversity and led to a depletion of anti-inflammatory matters and enrichment of proinflammatory factors [43] . A similar phenomenon was observed in the brains of 3xTg AD mice. ...
... A similar phenomenon was observed in the brains of 3xTg AD mice. Compared with the WT mice, the 3xTg mice also showed decreased abundance of Firmicutes and increased abundance of Bacteroidetes, which implies an increase in proinflammatory factors [43] . A previous study reported that 7,8-dihydroxyflavone (7,8-DHF), a small molecular agonist of TrkB, had a positive therapeutic effect in AD models [44] . ...
Alzheimer’s disease (AD) is the most common neurodegenerative disease, which is characterized by the deposition of senile plaques composed of amyloid-β (Aβ) and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau. Currently, the underlying cellular and molecular mechanisms of AD are still unclear. Growing evidence suggests that pathogen infections prominently promote the development of AD pathology. In this article, we reviewed the effect of multiple infectious pathogens that contribute to AD pathogenesis. Pathogens such as bacteria, viruses, and fungi are detected in the brains of AD patients and are known to be able to promote the development of AD pathology, including Aβ deposition and the formation of tau tangles. Here, we summarized the infectious pathogen-associated mechanisms of AD and provided new insight into the anti-infection remedy for AD.
... We used 50 μL dilution every 10 g of body weight per day. Fmr1 KO and WT control mice at P14 were administered 5 mg/kg 7,8-DHF [32] through intraperitoneal injections once daily for 16 consecutive days. To determine the appropriate dose, mice were weighted daily. ...
... Behavioral testing was conducted during the light phase of the circadian cycle, between 9.30 a.m. and 6.30 p.m. For in vitro experiments, primary neurons were treated with 7,8-DHF (0.5 μM, 1 μM, or 5 μM) at DIV 4 for 3 days [32]. The TrkB inhibitor K252a (50 nM or 100 nM) at DIV 6 for 24 h [32,33] or PLCγ agonist m-3M3FBS (25 μM) at DIV 5 for 3 h were used [34]. ...
... For in vitro experiments, primary neurons were treated with 7,8-DHF (0.5 μM, 1 μM, or 5 μM) at DIV 4 for 3 days [32]. The TrkB inhibitor K252a (50 nM or 100 nM) at DIV 6 for 24 h [32,33] or PLCγ agonist m-3M3FBS (25 μM) at DIV 5 for 3 h were used [34]. After treatment, Content courtesy of Springer Nature, terms of use apply. ...
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Fragile X syndrome (FXS) is the leading inherited form of intellectual disability and the most common cause of autism spectrum disorders. FXS patients exhibit severe syndromic features and behavioral alterations, including anxiety, hyperactivity, impulsivity, and aggression, in addition to cognitive impairment and seizures. At present, there are no effective treatments or cures for FXS. Previously, we have found the divergence of BDNF-TrkB signaling trajectories is associated with spine defects in early postnatal developmental stages of Fmr1 KO mice. Here, young fragile X mice were intraperitoneal injection with 7,8-Dihydroxyflavone (7,8-DHF), a high affinity tropomyosin receptor kinase B (TrkB) agonist. 7,8-DHF ameliorated morphological abnormities in dendritic spine and synaptic structure and rescued synaptic and hippocampus-dependent cognitive dysfunction. These observed improvements of 7,8-DHF involved decreased protein levels of BDNF, p-TrkBY816, p-PLCγ, and p-CaMKII in the hippocampus. In addition, 7,8-DHF intervention in primary hippocampal neurons increased p-TrkBY816 and activated the PLCγ1-CaMKII signaling pathway, leading to improvement of neuronal morphology. This study is the first to account for early life synaptic impairments, neuronal morphological, and cognitive delays in FXS in response to the abnormal BDNF-TrkB pathway. Present studies provide novel evidences about the effective early intervention in FXS mice at developmental stages and a strategy to produce powerful impacts on neural development, synaptic plasticity, and behaviors.
... Flavones, benzofurans, coumarins and quinolines are heterocyclic compounds with broad ranges of biological activities, such as antioxidant, anti-dementia and anti-inflammatory properties [23][24][25]. Although a few TRKB agonists such as flavone 7,8-DHF (7,8-dihydroxy flavone) have demonstrated neuroprotective potential against amyloid β (Aβ)-mediated neurodegeneration [26,27], the potential of TRKB agonists in Tau-mediated neurodegeneration remains to be determined. Previously, we found the effects of coumarin derivatives LMDS-1 and -2 against Tau-mediated neurotoxicity by activating TRKB signaling [8]. ...
... The neurite outgrowth promotion effects are attenuated by the knockdown of TRKB, which provides evidence that these compounds exert their beneficial effects through activating the TRKB signaling pathway. Furthermore, the tryptophan fluorescence assay demonstrates high binding affinities of ZN-015, VB-030 and VB-037 to TRKB-ECD, which are compatible with that of the reported TRKB agonist, 7,8-DHF [26,27]. ...
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Misfolded aggregation of the hyperphosphorylated microtubule binding protein Tau in the brain is a pathological hallmark of Alzheimer’s disease (AD). Tau aggregation downregulates brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB) signaling and leads to neurotoxicity. Therefore, enhancement of BDNF/TRKB signaling could be a strategy to alleviate Tau neurotoxicity. In this study, eight compounds were evaluated for the potential of inhibiting Tau misfolding in human neuroblastoma SH-SY5Y cells expressing the pro-aggregator Tau folding reporter (ΔK280 TauRD-DsRed). Among them, coumarin derivative ZN-015 and quinoline derivatives VB-030 and VB-037 displayed chemical chaperone activity to reduce ΔK280 TauRD aggregation and promote neurite outgrowth. Studies of TRKB signaling revealed that ZN-015, VB-030 and VB-037 treatments significantly increased phosphorylation of TRKB and downstream Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase 1/2 (ERK) and AKT serine/threonine kinase (AKT), to activate ribosomal S6 kinase (RSK) and cAMP response element-binding protein (CREB). Subsequently, p-CREB enhanced the transcription of pro-survival BDNF and BCL2 apoptosis regulator (BCL2), accompanied with reduced expression of anti-survival BCL2-associated X protein (BAX) in ΔK280 TauRD-DsRed-expressing cells. The neurite outgrowth promotion effect of ZN-015, VB-030 and VB-037 was counteracted by a RNA interference-mediated knockdown of TRKB, suggesting the role of these compounds acting as TRKB agonists. Tryptophan fluorescence quenching analysis showed that ZN-015, VB-030 and VB-037 interacted directly with a Pichia pastoris-expressed TRKB extracellular domain, indirectly supporting the role through TRKB signaling. The results of up-regulation in TRKB signaling open up the therapeutic potentials of ZN-015, VB-030 and VB-037 for AD.
... Tropo avin, also known as 7,8-Dihydroxy avone (7,, is a natural polyphenolic compound found in several vegetables, fruits, soy-based foodstuff, and tree leaves such as those of Godmania aesculifolia and Tridax procumbens [9,10]. For over 10 years, researchers have found and demonstrated that 7,8-DHF, a member of the avonoid family, has therapeutic e cacy in a variety of central nervous system disorders, including Alzheimer's disease [11,12], Parkinson's disease [13], Huntington's disease [14], and traumatic brain injury [15], and possesses anti-proliferative and anti-cancer effects in some cancer types [16,17]. Moreover, 7,8-DHF has been regarded as a powerful and selective anti-oxidative agent against tropomyosin receptor kinase B [18,19], oxidative stress-induced genotoxicity [20], and 6hydroxydopamine-induced dopaminergic cytotoxicity [21], and has been shown to protect against glutamate-induced excitotoxicity [22]. ...
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Malignant melanoma is one of the most progressive skin cancers, with a poor prognosis, various side effects, and high resistance to conventional treatment. Recently, 7,8-dihydroxyflavone (7,8-DHF), a polyphenolic flavonoid derived from certain plants, has been studied for its anti-carcinogenic, anti-inflammatory, antioxidant, and pharmacological effects in several types of cancer. Ganglioside, a modulator of diverse cell signals on the microdomain of the surface of the cell membrane, is closely involved in various cancers such as neuroblastoma. However, the correlation between ganglioside expression and the anti-cancer effects of 7,8-DHF in malignant melanoma remains unclear. In this study, 7,8-DHF showed potential as an anti-cancer agent through specific anti-proliferation, anti-oxidant, anti-migration, pro-apoptotic, and G2/M phase cell cycle arrest effects on SK-MEL-2 and G-361 melanoma cells. In contrast, 7,8-DHF did not induce cytotoxicity in non-tumoral epidermal HaCaT cells. Additionally, we confirmed for the first time that 7,8-DHF significantly reduces the expression levels of ganglioside GD3, which is closely involved in carcinogenesis, in both melanoma cancer cell lines using high-performance thin-layer chromatography analysis. Taken together, our findings suggest that 7,8-DHF might be a potent anticancer drug candidate for the treatment of malignant melanoma.
... Unfortunately, their actual therapeutic applications have been limited and the potential use of their beneficial effects remain to be exploited (Nagahara & Tuszynski, 2011). Neurotrophins, for example, have poor oral bioavailability, and very low stability in serum, with half-lives in the order of minutes (Zhang et al., 2014) as well as minimal BBB permeability and restricted diffusion within brain parenchyma (Pardridge, 2002). In addition, their receptor signaling networks can confer undesired off-target effects such as pain, spasticity and even neurodegeneration (Bergmann et al., 1998;Constandil et al., 2011;Dyck et al., 1997;Endo et al., 2009;Fahnestock et al., 2001;Fouad et al., 2013;Mufson et al., 2012). ...
The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA-12 only partly prevented the NDPIH-induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK-293T cells, an effect that was not sensitive to ANA-12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan-neurotrophic signalling pathway, leading to improved cognitive performance.
... For example, 7,8-dihydroxyflavone (7,8-DHF) displays high affinity to TRKB (Jang et al., 2010). Chronic oral administration of 7,8-DHF alleviates Aβ-induced synaptic loss and memory deficits in transgenic mice co-expressed five familial AD mutations (5×FAD mice) (Zhang et al., 2014). Although mimicking the physiological actions of BDNF and blood-brain barrier (BBB) permeable, 7,8-DHF has only moderate oral bioavailability and pharmacokinetic profile . ...
Alzheimer's disease (AD) is a neurodegenerative disease with progressive memory loss and the cognitive decline. AD is mainly caused by abnormal accumulation of misfolded amyloid β (Aβ), which leads to neurodegeneration via a number of possible mechanisms such as down-regulation of brain-derived neurotrophic factor-tropomyosin-related kinase B (BDNF-TRKB) signaling pathway. 7,8-Dihydroxyflavone (7,8-DHF), a TRKB agonist, has demonstrated potential to enhance BDNF-TRKB pathway in various neurodegenerative diseases. To expand the capacity of flavones as TRKB agonists, two natural flavones quercetin and apigenin, were evaluated. With tryptophan fluorescence quenching assay, we illustrated the direct interaction between quercetin/ apigenin and TRKB extracellular domain. Employing Aβ folding reporter SH-SY5Y cells, we showed that quercetin and apigenin reduced Aβ-aggregation, oxidative stress, caspase-1 and acetylcholinesterase activities, as well as improved the neurite outgrowth. Treatments with quercetin and apigenin increased TRKB Tyr516 and Tyr817 and downstream cAMP-response-element binding protein (CREB) Ser133 to activate transcription of BDNF and BCL2 apoptosis regulator (BCL2), as well as reduced the expression of pro-apoptotic BCL2 associated X protein (BAX). Knockdown of TRKB counteracted the improvement of neurite outgrowth by quercetin and apigenin. Our results demonstrate that quercetin and apigenin are to work likely as a direct agonist on TRKB for their neuroprotective action, strengthening the therapeutic potential of quercetin and apigenin in treating AD.
... Selective polyphenols also showed promising effects in rescuing cognitive function in transgenic AD mouse models. For example, old 5xFAD mice chronically treated with 7,8-dihydroxyflavone (7,8-DHF) reduced synapse loss in the brain and performed better at the working memory Y maze test (Devi and Ohno, 2012;Zhang et al., 2014). Oral administration of apigenin led to increased activation of ERK/CREB signaling and learning and memory improvement in 2xFAD mice (Zhao et al., 2013). ...
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Alzheimer′s disease (AD) is an irreversible progressive neurodegenerative disease affecting approximately 50 million people worldwide. It is estimated to reach 152 million by the year 2050. AD is the fifth leading cause of death among Americans age 65 and older. In spite of the significant burden the disease imposes upon patients, their families, our society, and our healthcare system, there is currently no cure for AD. The existing approved therapies only temporarily alleviate some of the disease’s symptoms, but are unable to modulate the onset and/or progression of the disease. Our failure in developing a cure for AD is attributable, in part, to the multifactorial complexity underlying AD pathophysiology. Nonetheless, the lack of successful pharmacological approaches has led to the consideration of alternative strategies that may help delay the onset and progression of AD. There is increasing recognition that certain dietary and nutrition factors may play important roles in protecting against select key AD pathologies. Consistent with this, select nutraceuticals and phytochemical compounds have demonstrated anti-amyloidogenic, antioxidative, anti-inflammatory, and neurotrophic properties and as such, could serve as lead candidates for further novel AD therapeutic developments. Here we summarize some of the more promising dietary phytochemicals, particularly polyphenols that have been shown to positively modulate some of the important AD pathogenesis aspects, such as reducing β-amyloid plaques and neurofibrillary tangles formation, AD-induced oxidative stress, neuroinflammation, and synapse loss. We also discuss the recent development of potential contribution of gut microbiome in dietary polyphenol function.
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Brain-derived neurotrophic factor (BDNF) is known to be involved in the pathogenesis of Alzheimers disease (AD). However, the pharmacological use of full-length neurotrophin is limited, because of its macromolecular protein nature. A dimeric dipeptide mimetic of the BDNF loop 1, bis-(N-monosuccinyl-L-methionyl-L-serine) heptamethylene diamide (GSB-214), was designed at the Zakusov Research Institute of Pharmacology. GSB-214 activates TrkB, PI3K/AKT, and PLC-1 in vitro. GSB-214 exhibited a neuroprotective activity during middle cerebral artery occlusion in rats when administered intraperitoneally (i.p.) at a dose of 0.1 mg/kg and improved memory in the novel object recognition test (0.1 and 1.0 mg/kg, i.p.). In the present study, we investigated the effects of GSB-214 on memory in the scopolamine- and steptozotocin-induced AD models, with reference to activation of TrkB receptors. AD was modeled in rats using a chronic i.p. scopolamine injection or a single streptozotocin injection into the cerebral ventricles. GSB-214 was administered within 10 days after the exposure to scopolamine at doses of 0.05, 0.1, and 1 mg/kg (i.p.) or within 14 days after the exposure to streptozotocin at a dose of 0.1 mg/kg (i.p.). The effect of the dipeptide was evaluated in the novel object recognition test; K252A, a selective inhibitor of tyrosine kinase receptors, was used to reveal a dependence between the mnemotropic action and Trk receptors. GSB-214 at doses of 0.05 and 0.1 mg/kg statistically significantly prevented scopolamine-induced long-term memory impairment, while not affecting short-term memory. In the streptozotocin-induced model, GSB-214 completely eliminated the impairment of short-term memory. No mnemotropic effect of GSB-214 was registered when Trk receptors were inhibited by K252A.
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Background: Alzheimer's disease (AD) is a multifactorial disorder characterized by cognitive decline. Current available therapeutics for AD have limited clinical benefit. Therefore, preventive therapies for interrupting the development of AD are critically needed. Molecules targeting multifunction to interact with various pathlogical components have been considered to improve the therapeutic efficiency of AD. In particular, herbal medicines with multiplicity of actions produce cognitive benefits on AD. Bugu-M is a multi-herbal extract composed of Ganoderma lucidum (Antler form), Nelumbo nucifera Gaertn., Ziziphus jujuba Mill., and Dimocarpus longan, with the ability of its various components to confer resilience to cognitive deficits. Objective: To evaluate the potential of Bugu-M on amyloid-β (Aβ) toxicity and its in vitro mechanisms and on in vivo cognitive function. Methods: We illustrated the effect of Bugu-M on Aβ25-35-evoked toxicity as well as its possible mechanisms to diminish the pathogenesis of AD in rat cortical neurons. For cognitive function studies, 2-month-old female 3×Tg-AD mice were administered 400 mg/kg Bugu-M for 30 days. Behavioral tests were performed to assess the efficacy of Bugu-M on cognitive impairment. Results: In primary cortical neuronal cultures, Bugu-M mitigated Aβ-evoked toxicity by reducing cytoskeletal aberrations and axonal disruption, restoring presynaptic and postsynaptic protein expression, suppressing mitochondrial damage and apoptotic signaling, and reserving neurogenic and neurotrophic factors. Importantly, 30-day administration of Bugu-M effectively prevented development of cognitive impairment in 3-month-old female 3×Tg-AD mice. Conclusion: Bugu-M might be beneficial in delaying the progression of AD, and thus warrants consideration for its preventive potential for AD.
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The neuropathological correlates of Alzheimer's disease (AD) include amyloid-β (Aβ) plaques and neurofibrillary tangles. To study the interaction between Aβ and tau and their effect on synaptic function, we derived a triple-transgenic model (3×Tg-AD) harboring PS1M146V, APPSwe, and tauP301L transgenes. Rather than crossing independent lines, we microinjected two transgenes into single-cell embryos from homozygous PS1M146V knockin mice, generating mice with the same genetic background. 3×Tg-AD mice progressively develop plaques and tangles. Synaptic dysfunction, including LTP deficits, manifests in an age-related manner, but before plaque and tangle pathology. Deficits in long-term synaptic plasticity correlate with the accumulation of intraneuronal Aβ. These studies suggest a novel pathogenic role for intraneuronal Aβ with regards to synaptic plasticity. The recapitulation of salient features of AD in these mice clarifies the relationships between Aβ, synaptic dysfunction, and tangles and provides a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be assessed.
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The aggregation of the 42-residue amyloid β-protein (Aβ42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aβ42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3′,4′-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aβ42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aβ42-taxifolin adduct was suggested by mass spectrometry, Aβ42 mutants substituted at Arg⁵, Lys¹⁶, and/or Lys²⁸ with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aβ42 mutants at Lys¹⁶ and/or Lys²⁸ except for the mutant at Arg⁵. In addition, the aggregation of Aβ42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aβ42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aβ42 as well as Aβ42. Furthermore, interaction of (+)-taxifolin with the β-sheet region in Aβ42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aβ42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aβ42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy. Background: The inhibitory mechanism of Aβ42 aggregation by flavonoid is fully unknown. Results: The oxidant enhanced the inhibitory activity of (+)-taxifolin against Aβ42 aggregation by forming Aβ42-taxifolin adducts between the Lys residues and oxidized (+)-taxifolin. Conclusion: The inhibitory activity of catechol-type flavonoids requires autoxidation to form an o-quinone to react with Lys. Significance: These may help design promising inhibitors against Aβ42 aggregation for Alzheimer disease therapy.
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Increasing evidence suggests that synaptic dysfunction is a key pathophysiological hallmark in neurodegenerative disorders, including Alzheimer's disease. Understanding the role of brain-derived neurotrophic factor (BDNF) in synaptic plasticity and synaptogenesis, the impact of the BDNF Val66Met polymorphism in Alzheimer's disease-relevant endophenotypes - including episodic memory and hippocampal volume - and the technological progress in measuring synaptic changes in humans all pave the way for a 'synaptic repair' therapy for neurodegenerative diseases that targets pathophysiology rather than pathogenesis. This article reviews the key issues in translating BDNF biology into synaptic repair therapies.
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Dendritic spines are major sites of excitatory synaptic transmission and changes in their numbers and morphology have been associated with neurodevelopmental and neurodegenerative disorders. Brain-derived Neurotrophic Factor (BDNF) is a secreted growth factor that influences hippocampal, striatal and neocortical pyramidal neuron dendritic spine density. However, the mechanisms by which BDNF regulates dendritic spines, and how BDNF interacts with other regulators of spines remain unclear. We propose that one mechanism by which BDNF promotes dendritic spine formation is through an interaction with Wnt signaling. Here, we show that Wnt signaling inhibition in cultured cortical neurons disrupts dendritic spine development, reduces dendritic arbor size and complexity, and blocks BDNF-induced dendritic spine formation and maturation. Additionally, we show that BDNF regulates expression of Wnt2, and that Wnt2 is sufficient to promote cortical dendrite growth and dendritic spine formation. Together, these data suggest that BDNF and Wnt signaling cooperatively regulate dendritic spine formation.
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7,8-Dihydroxyflavone (7,8-DHF) acts as a TrkB receptor-specific agonist. It mimics the physiological actions of brain-derived neurotrophic factor (BDNF) and demonstrates remarkable therapeutic efficacy in animal models of various neurological diseases. Nonetheless, its in vivo pharmacokinetic profiles and metabolism remain unclear. Here we report that 7,8-DHF and its O-methylated metabolites distribute in mouse brain after oral administration. Both hydroxy groups can be mono-methylated, and the mono-methylated metabolites activate TrkB in vitro and in vivo. Blocking methylation, using COMT inhibitors, diminishes the agonistic effect of TrkB activation by 7,8-DHF or 4'-dimethylamino-7,8-DHF, supporting the contribution of the methylated metabolite to TrkB activation in mouse brain. Moreover, we have synthesized several methylated metabolite derivatives, and they also potently activate the TrkB receptor and reduce immobility in both forced swim test and tail suspension test, indicating that these methylated metabolites may possess antidepressant activity. Hence, our data demonstrate that 7,8-DHF is orally bioavailable and can penetrate the brain-blood barrier. The O-methylated metabolites are implicated in TrkB receptor activation in the brain.
USING two-site enzyme immunoassays (EIAs), we measured the levels of neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) simultaneously in three brain regions (motor cortex, dentate gyrus and entorhinal cortex) of patients with Alzheimer's disease (AD) and control individuals. Significant differences between the neurotrophin levels of these two groups were found in the different brain regions depending on the neurotrophin. The NGF level in the dentate gyrus of AD patients was higher, whereas the BDNF level in the entorhinal cortex and the NT-3 level in the motor cortex were lower than the corresponding control levels. These results indicate that protein levels of individual neurotrophins in different brain regions are affected differently by AD, and such differential changes may contribute to the complex pathology of AD.
Brain-derived neurotrophic factor (BDNF) modulates the synaptic transmission of several monoaminergic neuronal systems. Molecular techniques using synapatosomes in previous studies have suggested that BDNF's receptor, tyrosine kinases (Trk), can quickly regulate dopamine release and transporter dynamics. Our main objective in this study is to determine whether slice fast scan cyclic voltammetry can be used to investigate the role of the TrkB receptor on dopamine release and uptake processes in the caudate-putamen. Fast scan cyclic voltammetry measured dopamine release and uptake rates in the presence of BDNF, or its agonist 7,8-dihydroxyflavone, or a TrkB inhibitor K252a. Superfusion of BDNF led to partial recovery of the electrically-stimulated dopamine release response in BDNF+/- mice which is blunted compared to wildtype mice, with no effect in wildtype mice. Conversely, infusion of 7,8-dihydroxyflavone increased electrically-stimulated dopamine release in wildtype mice with no difference in BDNF+/- mice. Overall, BDNF and 7,8-dihydroxyflavone had no effect on dopamine uptake rates. Concentrations greater than 3 μM 7,8-dihydroxyflavone affected DA uptake rates in BDNF+/- mice only. To demonstrate that BDNF and 7,8-dihydroxyflavone modulate DA release by activating the TrkB receptor, both genotypes were pretreated with K252a. K252a was able to block BDNF and 7,8-DHF induced increases during stimulated dopamine release in BDNF+/- and wildtype mice, respectively. Fast scan cyclic voltammetry demonstrates that acute TrkB activation potentiates dopamine release in both genotypes.
The Alzheimer's disease (AD) epidemic is a looming crisis, with an urgent need for new therapies to delay or prevent symptom onset and progression. There is growing awareness that clinical trials must target stage-appropriate pathophysiological mechanisms to effectively develop disease-modifying treatments. Advances in AD biomarker research have demonstrated changes in amyloid-beta (Aβ), brain metabolism and other pathophysiologies prior to the onset of memory loss, with some markers possibly changing one or two decades earlier. These findings suggest that amyloid-based therapies would optimally be targeted at the earliest clinically detectable stage (such as mild cognitive impairment (MCI)) or before. Postmortem data indicate that tau lesions in the locus coeruleus (LC), the primary source of subcortical norepinephrine (NE), may be the first identifiable pathology of AD, and recent data from basic research in animal models of AD indicate that loss of NE incites a neurotoxic proinflammatory condition, reduces Aβ clearance and negatively impacts cognition - recapitulating key aspects of AD. In addition, evidence linking NE deficiency to neuroinflammation in AD also exists. By promoting proinflammatory responses, suppressing anti-inflammatory responses and impairing Aβ degradation and clearance, LC degeneration and NE loss can be considered a triple threat to AD pathogenesis. Remarkably, restoration of NE reverses these effects and slows neurodegeneration in animal models, raising the possibility that treatments which increase NE transmission may have the potential to delay or reverse AD-related pathology. This review describes the evidence supporting a key role for noradrenergic-based therapies to slow or prevent progressive neurodegeneration in AD. Specifically, since MCI coincides with the onset of clinical symptoms and brain atrophy, and LC pathology is already present at this early stage of AD pathogenesis, MCI may offer a critical window of time to initiate novel noradrenergic-based therapies aimed at the secondary wave of events that lead to progressive neurodegeneration. Because of the widespread clinical use of drugs with a NE-based mechanism of action, there are immediate opportunities to repurpose existing medications. For example, NE transport inhibitors and NE-precursor therapies that are used for treatment of neurologic and psychiatric disorders have shown promise in animal models of AD, and are now prime candidates for early-phase clinical trials in humans.