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\begin{document}

\title{Dysfunction of the BDNF-TrkB signaling pathway contributes to learning
and memory impairments induced by neuroinflammation in mice}



\author[1]{Wen Zhang}%
\author[2]{Mengmeng Ge}%
\author[3]{ Long-qing Zhang}%
\author[1]{Xiaoman Yuan}%
\author[1]{Siyi Han}%
\author[4]{Yuke Tian}%
\author[3]{Xue-Bi  Tian}%
\affil[1]{Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology}%
\affil[2]{Department of Anesthesiology and Pain Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology}%
\affil[3]{Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology}%
\affil[4]{Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose: Evidence suggests neuroinflammation is the main
mechanism in cognitive dysfunction. The brain-derived neurotrophic
factor (BDNF) is involved in learning and memory via binding to tyrosine
kinase B (TrkB) receptors. Herein, we mainly tested roles of the
BDNF-TrkB signaling pathway and its downstream cascades in
lipopolysaccharide (LPS) induced cognitive dysfunction in mice.
Experimental Approach: Mice were treated with LPS and 7,8-DHF for 7
days, and learning and memory function was evaluated by the novel object
recognition test (NORT). Western blot and enzyme-linked immunosorbent
assay (ELISA) elucidated roles of the BDNF-TrkB signaling pathway and
its downstream cascades in LPS mice. Key Results: The NORT showed that
LPS induced learning and memory deficits in mice. LPS increased the
levels of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α \selectlanguage{english}in the serum of mice. In the
hippocampus and mPFC regions, LPS reduced protein levels of BDNF,
p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 and increased the
expression of Bax. In the EC, levels of BDNF, p-TrkB, Bcl-2, p-CaMK2 and
p-CREB proteins were reduced and the protein level of Bax was increased
in LPS mice. 7,8-DHF could alleviate these disorders in LPS mice and
improved their learning and memory function, however, the TrkB
antagonist ANA12 effectively reversed the effects of 7,8-DHF. Conclusion
and Implications: These results revealed that the BDNF-TrkB signaling
pathway and its downstream cascades disorders contributed to
neuroinflammation induced cognitive dysfunction in mice. 7,8-DHF could
become a new therapeutic drug for cognitive dysfunction induced by
BDNF-TrkB signaling pathway disorders in neurodegenerative diseases.%
\end{abstract}%



\sloppy


\textbf{Dysfunction of the BDNF-TrkB signaling pathway contributes to
learning and memory impairments induced by neuroinflammation in mice}

Running title: Role of the BDNF-TrkB signaling pathway in cognitive
dysfunction

\textbf{Authors:}

Wen Zhang\textsuperscript{1}, Meng-meng Ge\textsuperscript{1}, Long-Qing
Zhang\textsuperscript{1}, Xiao-Man Yuan\textsuperscript{1}, Si-yi
Han\textsuperscript{1}, Anne Manyande\textsuperscript{3}, Xue-Bi
Tian\textsuperscript{1}*, Yu-Ke Tian\textsuperscript{1}*

\textbf{Affiliations}

1. Department of Anesthesiology, Tongji Hospital, Tongji Medical
College, Huazhong University of Science and Technology, Wuhan, Hubei,
China

2. School of Human and Social Sciences, University of West London,
London, UK

\textbf{*Corresponding authors} :

Xue-Bi Tian, PhD

Department of Anesthesiology, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, Hubei, China.

E-mail: tianxb@hust.edu.cn

Yu-Ke Tian, PhD

Department of Anesthesiology, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, Hubei, China.

E-mail:\emph{yktian@tjh.tjmu.edu.cn}

Tel: +011862783663423;

Fax: +011862783662853;

Word count (excluding abstract, methods, figure legends and references):
2905

Figure: 9

\textbf{Bullet point summary}

What is already known

\begin{enumerate}
\tightlist
\item
  The BDNF-TrkB signaling pathway plays a significant role in learning
  and memory impairment.
\item
  ERK1/2, CaMK2, CREB, GluR1, and apoptosis are involved in learning and
  memory procession.
\item
  7,8-DHF is an effective agonist of the TrkB receptor which mimics the
  properties of BDNF.
\end{enumerate}

What this study adds

\begin{enumerate}
\tightlist
\item
  The BDNF-TrkB signaling pathway and its downstream cascades disorders
  involved in the LPS-induced learning and memory impairment in mice.
\item
  7,8-DHF could effectively alleviate cognitive dysfunction via acting
  on the TrkB in LPS mice.
\item
  The mPFC, hippocampus and EC regions are associated with learning and
  memory procession.
\end{enumerate}

Clinical significance

The BDNF-TrkB signaling pathway and its downstream cascade disorders are
a new viewpoint for learning and memory impairments induced by
neuroinflammation.

7,8-DHF might serve as a potential target for preventing or treating
cognitive dysfunction induced by neuroinflammation in neurodegenerative
diseases.

\textbf{Acknowledgments}

This work was financially supported by grants from the National Natural
Science Foundation of China 81571053 and 81974170.

\textbf{Data Availability Statement}

Data available on request from the authors. The data that support the
findings of this study are available from the corresponding authors upon
reasonable request. Some data may not be made available because of
privacy or ethical restrictions.

\textbf{Abbreviations:}

BDNF: brain-derived neurotrophic factor

7,8-DHF: 7,8- Dihydroxyflavone

ELISA: enzyme-linked immunosorbent assay

LPS: lipopolysaccharide

NORT: novel object recognition test

p75NTR: p75 neurotrophin receptor

TrkB: tyrosine kinase B

\textbf{Abstract}

\textbf{Background and Purpose:}Evidence suggests neuroinflammation is
the main mechanism in cognitive dysfunction. The brain-derived
neurotrophic factor (BDNF) is involved in learning and memory via
binding to tyrosine kinase B (TrkB) receptors. Herein, we mainly tested
roles of the BDNF-TrkB signaling pathway and its downstream cascades in
lipopolysaccharide (LPS) induced cognitive dysfunction in mice.

\textbf{Experimental Approach:} Mice were treated with LPS and 7,8-DHF
for 7 days, and learning and memory function was evaluated by the novel
object recognition test (NORT). Western blot and enzyme-linked
immunosorbent assay (ELISA) elucidated roles of the BDNF-TrkB signaling
pathway and its downstream cascades in LPS mice.

\textbf{Key Results:} The NORT showed that LPS induced learning and
memory deficits in mice. LPS increased the levels of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and
TNF-\selectlanguage{greek}α \selectlanguage{english}in the serum of mice. In the hippocampus and mPFC regions, LPS
reduced protein levels of BDNF, p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB
and p-GluR1 and increased the expression of Bax. In the EC, levels of
BDNF, p-TrkB, Bcl-2, p-CaMK2 and p-CREB proteins were reduced and the
protein level of Bax was increased in LPS mice. 7,8-DHF could alleviate
these disorders in LPS mice and improved their learning and memory
function, however, the TrkB antagonist ANA12 effectively reversed the
effects of 7,8-DHF.

\textbf{Conclusion and Implications:} These results revealed that the
BDNF-TrkB signaling pathway and its downstream cascades disorders
contributed to neuroinflammation induced cognitive dysfunction in mice.
7,8-DHF could become a new therapeutic drug for cognitive dysfunction
induced by BDNF-TrkB signaling pathway disorders in neurodegenerative
diseases.

\textbf{Keywords}

7,8-Dihydroxyflavone, Lipopolysaccharide, Brain-derived neurotrophic
factor, Tyrosine kinase B, Learning and memory impairment

\textbf{Introduction}

Systemic inflammation often promotes central neuroinflammation by
up-regulating pro-inflammatory cytokines, increasing the permeability of
the blood-brain barrier and further causing neuroinflammation in the
central nervous system(Adetuyi \& Farombi, 2021; Zhan, Stamova, \&
Sharp, 2018), which further activates the inflammatory signaling
pathway, that triggers synaptic plasticity changes and neuronal
apoptosis , ultimately leading to cognitive dysfunction and
neurodegenerative diseases(Li et al., 2020; H. Zhang et al., 2020; Zhao
et al., 2019). For example, systemic infection which occurs with
surgery(Cibelli et al., 2010; Wan et al., 2007), can trigger systemic
and hippocampal inflammation resulting in cognitive decline(Barrientos
et al., 2009).

The brain-derived neurotrophic factor (BDNF) is the main neurotrophin
growth factor that participates in mediating synaptic plasticity and
neuronal survival, differentiation, as well as neurogenesis(Leal,
Bramham, \& Duarte, 2017; von Bohlen und Halbach \& von Bohlen und
Halbach, 2018). BDNF exerts its biological functions mainly through
binding to tyrosine kinase B (TrkB) receptors(Bekinschtein, Cammarota,
\& Medina, 2014; Lu, Nagappan, \& Lu, 2014) and the BDNF-TrkB signaling
pathway is involved in the formation of dendritic spines. Furthermore,
activation of TrkB receptors promotes activations of downstream cascades
including the ERK1/2, CaMK2, CREB, GluR1 and the mechanism of apoptosis
which are related to regulating learning and memory(Y. J. Yang et al.,
2014). The ERK1/2, a member of the mitogen-activated protein kinase
superfamily, is involved in regulating cell proliferation, survival, and
apoptosis(Morella, Hallum, \& Brambilla, 2020). The CaMK2 is essential
for synaptic plasticity and memory formation(Kool et al., 2019). The
CREB, a transcription factor, is one of the main downstream
transcription factors of ERK1/2 and plays significant roles in neuronal
plasticity, learning and memory(B. Zhang et al., 2020). The GluR1 is
also necessary for learning and memory processing and mediates neuronal
plasticity(Xiao, Liu, Li, Ruan, \& Wang, 2019). Therefore, the reduction
of BDNF expression in the brain is associated with impairment of
synaptic plasticity and learning and memory failures.

7,8- Dihydroxyflavone (7,8-DHF), a member of the flavonoid derivative,
has been identified as an effective agonist of the TrkB receptor which
mimics the properties of BDNF(Bollen et al., 2013; Castello et al.,
2014; Zhang et al., 2014). 7,8-DHF can penetrate the blood-brain barrier
and plays neuroprotective roles including increasing dendritic spine
density and exerting neurotrophic effects, via activating the BDNF-TrkB
signaling pathway and its downstream cascades. ANA12 is a small molecule
antagonist of TrkB and crosses the blood-brain barrier in order to exert
its antagonism effect(Giuliani, 2019). In the current research, we
mainly investigated the roles of the BDNF-TrkB signaling pathway and its
downstream cascades on neuroinflammation-induced learning and memory
impairments and whether 7,8-DHF could alleviate these disorders in mice.

\textbf{Materials and Methods}

\textbf{Animals}

Male c57BL/6J mice (8 weeks old) were obtained from the Experimental
Animal Center of Tongji Medical College, Huazhong University of Science
and Technology. A total of 106 mice were used in our research. Mice were
raised five per cage under 12 hours light/dark cycle and given water and
food ad libitum. All animal studies were approved by the Experimental
Animal Committee of Tongji Medical college and in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals.

\textbf{Experimental design}

Mice were divided into four groups: Control group, LPS group,
LPS+7,8-DHF group and LPS+7,8-DHF+ANA12 group. In the LPS group, mice
were intraperitoneally injected with LPS at a concentration of 0.25
mg/kg. In the Control group, mice were intraperitoneally injected with
the same amount of saline. In the LPS+7,8-DHF group, 7,8-DHF (5 mg/kg)
were injected 30 minutes before LPS treatment. And in the
LPS+7,8-DHF+ANA12 group, ANA12 (0.5 mg/kg) were injected 30 minutes
before 7,8-DHF and LPS. The novel object recognition test was performed
to detect cognitive function 7 days after treatment.

\textbf{Novel object recognition test}

7 days after LPS treatment, the novel object recognition test (NORT) was
applied to examine cognitive function in mice(W. Zhang et al., 2020).
Mice were handled 6 days before the test, for 1 minute a day. Then,
putting mice into the box for 5 minutes to acclimatization. 24 hours
later, mice were allowed to explore two identical rectangular blocks for
5 minutes. After 2 hours, a cylinder was used to replace one of the
rectangular blocks, and mice explored for another 5 minutes. Exploratory
behaviors were performed as sniff, lick and climb. Discrimination ratio
was used to assess learning and memory.

\textbf{Enzyme-linked immunosorbent assay (ELISA)}

Serum samples were collected and centrifuged for 10 minutes at 3000g at
4 \selectlanguage{ngerman}°C to obtain supernatants. Expressions of IL-6, IL-1\selectlanguage{greek}β, \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}were
determined using commercially available ELISA kits (MDL Biotech,
Beijing, China). Concentrations of inflammation factors were tested
according to the manufacturer's instructions and presented as pg/ml of
serum protein.

\textbf{Western bolt}

Hippocampal tissues were prepared as previously described(W. Zhang et
al., 2020) and homogenized with radioimmunoprecipitation assay lysis
buffer containing protease and phosphatase inhibitors. The commercially
available BCA protein assay kit (Boster, Wuhan, China) was used to
determine protein concentrations. Then, protein samples were separated
by 10\% SDS-PAGE and transferred to polyvinylidene fluoride membranes
(Millipore, Bedford, MA, USA). Bands were blocked with 5\% BSA for 1.5
hours at room temperature. Primary antibodies were incubated overnight
at 4 \selectlanguage{ngerman}°C, and then, bands were washed with TBST and incubated with
horseradish peroxidase-conjugated secondary antibodies at room
temperature for 2 hours. The antibodies used in this research include
rabbit anti-BDNF (1:1000; Abclonal), anti-TrkB (1:1000; Abclonal),
anti-p-TrkB (1:500; Abclonal), anti-Bax (1:1000; Abclonal), anti-Bcl-2
(1:1000; Abclonal), anti-ERK1/2 (1:1000; Cell Signaling Technology),
anti-p-ERK1/2 (1:1000; Cell Signaling Technology), anti-CaMK2 (1:1000;
Proteintech), anti-p-CaMK2 (1:500; Abclonal), anti-CREB (1:1000;
Abclonal), anti-p-CREB (1:1000; Cell Signaling Technology), anti-GluR1
(1:1000; Abclonal), anti-p-GluR1 (1:500; Abclonal), anti-GAPDH (1:1000;
Abclonal), and goat anti-rabbit (1:5000; Promoter). The bands were
visualized using chemiluminescence (Pierce ECL Western Blotting
Substrate, Abbkine) and measured by a computerized image analysis system
(ChemiDoc XRS+, BIO-RAD, CA, USA).

\textbf{Statistical analysis}

All data were analyzed using GraphPad prism version 7.0 and presented as
mean ± SEM. For two groups, unpaired Student's \emph{t} test was
applied. For multiple groups, One-way ANOVA was used. In addition,
two-way ANOVA was used to analyze data of the time spent with the object
in the novel object recognition test. \emph{P} \textless{} 0.05 was
considered statistically significant.

\textbf{Results}

\textbf{Lipopolysaccharide resulted inlearning and memory impairment in
mice}

The NORT is a widely used to evaluate learning and memory function in
mice(W. Zhang et al., 2020; Zurek, Bridgwater, \& Orser, 2012). In this
study, no significant difference was found in time spent with objects
between the Control and LPS mice (Figure 1A). In the testing stage,
spent more time was spent on the novel object than on the familiar
object in the Control mice (Figure 1B), but there was no statistic
difference in LPS mice (Figure 1B). The result of discrimination ratio
was similar to that in Figure 1B which shows that Control mice had
greater discrimination ratio than LPS mice (Figure 1C). Then, we
extracted serum to detect the concentrations of IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α
\selectlanguage{english}after LPS treatment. The result indicates that levels of IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and
TNF-\selectlanguage{greek}α \selectlanguage{english}were increased in the LPS group (Figure 1D). These findings
demonstrate that the administration of LPS resulted in systemic
inflammation that induced learning and memory impairment in mice.

\textbf{Lipopolysaccharide disturbed the BDNF-TrkB signaling pathway and
its downstream cascades in the hippocampus}

After the novel object recognition test, we segregated the hippocampus
to detect protein levels of BDNF-TrkB signaling using western blot. The
results show that, compared with the Control group, the protein level of
BDNF in the hippocampus was reduced in LPS mice (Figure 2A-B). No
statistical difference was found in TrkB protein levels between the
Control and LPS mice (Figure 2A, C). However, the protein level of the
p-TrkB was reduced in LPS mice (Figure 2A, D). Then, we detected levels
of apoptosis-related proteins. Compared with the Control mice, the level
of pro-apoptotic protein Bax was distinctly increased, while the level
of anti-apoptotic protein Bcl-2 was decreased in LPS mice (Figure 2
E-G). In addition, phosphorylation protein levels of TrkB downstream
cascades were tested. The protein levels of p-ERK1/2, p-CaMK2, p-CREB
and p-GluR1were obviously reduced in LPS mice (Figure 2H, J-K, M-N, P-Q,
S). However, no difference was found in levels of ERK1/2, CaMK2, CREB
and GluR1 proteins (Figure 2H-I, K-L, N-O, Q-R). These results
illustrate that the BDNF-TrkB signaling pathway and its downstream
cascades were regulated by inflammation and may be involved in learning
and memory processes.

\textbf{Lipopolysaccharide disturbed the B} \textbf{DNF-TrkB signaling
pathway and its downstream cascades in the mPFC}

Then, we tested the protein levels of the BDNF-TrkB signaling pathway
and its downstream cascades in the mPFC. The western blot results
revealed that the levels of BDNF and p-TrkB were reduced after
administration of LPS (Figure 3A-B, D), and no difference was found in
the protein level of TrkB (Figure 3A, C). Compared to the Control group,
the expression of Bax was up-regulated in LPS mice (Figure 3E-F). At the
same time, reduction of Bcl-2 expression was found in the LPS group
(Figure 3E, G). Furthermore, the expression of p-ERK1/2, p-CaMK2, p-CREB
and p-GluR1 proteins was decreased markedly in LPS mice (Figure 3H, J-K,
M-N, P-Q, S). However, there were no significant differences in the
expression of ERK1/2, CaMK2, CREB and GluR1 proteins (Figure 3H-I, K-L,
N-O, Q-R). These results demonstrate that the BDNF-TrkB signaling
pathway and its downstream cascades in the mPFC play a vital role in
learning and memory processes.

\textbf{Lipopolysaccharide disturbed the BDNF-TrkB signaling pathway and
its downstream cascades in the EC}

Next, the protein levels of the BDNF-TrkB signaling pathway and its
downstream cascades in the EC were also examined using western blot. The
results show that a qualitative reduction in the expression of BDNF and
p-TrkB was observed in LPS mice (Figure 4A-B, D). There was no
difference in the levels of TrkB protein between Control and LPS groups
(Figure 4A, C). In addition, the protein level of Bax was increased
compared to Control mice (Figure 4E-F), and the expression of Bcl-2 was
markedly decreased in LPS mice (Figure 4E, G). No statistical difference
was observed in protein levels of ERK1/2, p-ERK1/2, GluR1 and p-GluR1
between Control and LPS mice (Figure 4H-J, Q-S). The expression of
p-CaMK2 and p-CREB were obviously reduced after using LPS (Figure 4K,
M-N, P). No difference was shown in levels of CaMK2 and CREB (Figure
4K-L, N-O). These results suggest that, in the EC, the BDNF-TrkB
signaling pathway, p-CaMK2 and p-CREB cascades participate in process of
learning and memory.

\textbf{7,8-DHF ameliorated lipopolysaccharide induced learning and
memory deficits in mice}

7,8-DHF was used to detect the role of the BDNF-TrkB signaling pathway
in learning and memory(Bollen et al., 2013). ANA12 is a small-molecule
TrkB antagonist(Cazorla et al., 2011), which was used to identify
whether 7,8-DHF could reverse learning and memory dysfunction. The
results of the novel object recognition test shows that there were no
statistical changes in the time spent with identical objects during the
training stage (Figure 5A). In the testing stage, administration of
7,8-DHF increased the time spent on the novel object in LPS mice, but
ANA12 reversed the role of 7,8-DHF in LPS mice (Figure 5B). Further
analysis of the discrimination ratio was in line with the result of time
spent with an object. 7,8-DHF up-regulated the discrimination ratio in
LPS+7,8-DHF mice, however, the discrimination ratio was reduced in
LPS+7,8-DHF+ANA12 mice (Figure 5C). These results demonstrate that the
preventive use of 7,8-DHF could effectively alleviate the dysfunction of
learning and memory caused by LPS, while ANA12 reversed 7,8-DHF
therapeutical effects.

\textbf{7,8-DHF ameliorated lipopolysaccharide induced the BDNF-TrkB
signaling pathway and its downstream cascade disorders in the
hippocampus}

The hippocampus was observed to test changes in protein levels after
administration of 7,8-HDF. Western blot results show that 7,8-DHF
up-regulated the expression of p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB,
p-GluR1, and decreased the protein level of Bax in LPS mice (Figure 6A,
D-H, J-K, M-N, P-Q, S). But ANA12 reversed these changes in 7.8-DHF of
LPS+7,8-DHF+ANA12 mice. There was no significant increase in expression
of BDNF after using 7,8-DHF or reduction in the LPS+7,8-DHF+ANA12 group
(Figure 6A-B). In addition, there were no significant differences in the
expression of TrkB, ERK1/2, CaMK2, CREB and GluR1(Figure 6A, C, H-I,
K-L, N-O, Q-R). These results demonstrate that 7,8-DHF effectively
alleviated disorders of the BDNF-TrkB signaling pathway and its
downstream cascades in the hippocampus of LPS mice, but ANA12 completely
reversed the therapeutic effects of 7,8-DHF.

\textbf{7,8-DHF ameliorated the lipopolysaccharide induced BDNF-TrkB
signaling pathway and its downstream cascade disorders in the mPFC}

Furthermore, we tested protein levels of the BDNF-TrkB signaling pathway
and its downstream cascades in the mPFC after administration of 7,8-DHF
using western blot.

The results are consistent with hippocampal data. The protein levels of
p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB, p-GluR1 were increased, and
the expression of Bax was reduced in the LPS+7,8-DHF group (Figure 7A,
D-H, J-K, M-N, P-Q, S). But the effects of 7,8-DHF were reversed by
ANA12 in LPS+7,8-DHF+ANA12 mice. There were no changes in the level of
BDNF after using 7,8-DHF or 7,8-DHF+ANA12 in LPS mice (Figure 7A-B).
Furthermore, no statistical difference was observed in the expression of
TrkB, ERK1/2, CaMK2, CREB and GluR1(Figure 7A, C, H-I, K-L, N-O, Q-R).
These results confirm that the disorders of the BDNF-TrkB signaling
pathway and its downstream cascades in the mPFC are alleviated using
7,8-DHF in LPS mice, but ANA12 antagonized the protective effects of
7,8-DHF.

\textbf{7,8-DHF ameliorated lipopolysaccharide induced the BDNF-TrkB
signaling pathway and its downstream cascade disorders in the EC}

Finally, the expression changes in related proteins of the EC were
detected by western blot. The results show that a qualitative increase
in p-TrkB, Bcl-2, p-CaMK2, p-CREB expression and reduction in Bax
expression was observed in LPS+7,8-DHF mice (Figure 8A, D-G, KC, M-N,
P). However, the effects of 7,8-DHF were reversed by ANA12 in
LPS+7,8-DHF+ANA12 mice. Consistent with previous results, 7,8-DHF and
ANA12 had no effect on the expression of BDNF protein (Figure 8A-B). In
addition, there were no significant differences in the expression of
TrkB, ERK1/2, p-ERK1/2, CaMK2, CREB, GluR1 and p-GluR1 proteins (Figure
8A, C, H-L, N-O, Q-S). These results signify that 7,8-DHF can
effectively alleviate disorders of the BDNF-TrkB signaling pathway,
p-CaMK2 and p-CREB cascades in the EC of LPS mice, but ANA12 can
antagonize the therapeutical effects of 7,8-DHF.

\textbf{Discussion and conclusions}

Numerous studies have shown that the central neuroinflammatory response
is the main mechanism of neurodegenerative diseases, but the specific
pathophysiological mechanism is still unclear. In this study, we found
that disorders of the BDNF-TrkB signaling pathway and its downstream
cascade in mice participated in learning and memory impairments induced
by neuroinflammation, which mainly manifested as reductions in BDNF,
p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 proteins and
upregulation of the expression of Bax protein in different brain regions
including the mPFC, hippocampus and EC.

It is widely known that BDNF is the main neurotrophin in the brain(von
Bohlen und Halbach \& von Bohlen und Halbach, 2018). Many researchers
have reported that BDNF has important effects on synapses including
structural and functional roles in many brain regions(Bekinschtein et
al., 2014; Lu et al., 2014). The different effects of BDNF on the brain
are related to its diverse forms and downstream receptors that it binds
to, such as pro-BDNF and mature-BDNF with the p75 neurotrophin receptor
(p75NTR) and TrkB which have opposing effects on synapses(Fobian et al.,
2009; Kowia\selectlanguage{polish}ń\selectlanguage{english}ski et al., 2017; Sasi, Vignoli, Canossa, \& Blum, 2017). In
our study, we mainly discuss the roles of mature-BDNF and TrkB receptors
on learning and memory deficits induced by neuroinflammation. The
expression of BDNF and p-TrkB receptors were reduced in the mPFC,
hippocampus and EC regions after application of LPS in mice. This
demonstrates that different ways of applying BDNF induced different
changes in functions and structures(Ji et al., 2010). For example, acute
up-regulation of BDNF initiated neurite elongation and spine head
enlargement, however, gradual increase in BDNF potentiated dendritic
branching and filopodia-like spines. In addition, transient activation
of TrkB promoted synaptic transmission in the brain. However, systematic
application of BDNF could be catabolized by enzymes in vivo. In this
study, we used TrkB agonist 7,8-DHF to simulate physiological actions of
BDNF and detected whether the activation of TrkB could alleviate
cognitive impairments in LPS mice. As the results show, preventive use
of 7,8-DHF effectively alleviated learning and memory dysfunction
induced by LPS. At the same time, the level of p-TrkB was increased
after administration of 7,8-DHF. However, the application of TrkB
antagonist ANA12 completely reversed therapeutic effects of 7,8-DHF,
which further indicates that the BDNF-TrkB signaling pathway disorder
involved neuroinflammation associated with learning and memory
impairments in mice.

Increasing evidence suggests that BDNF exerts its neuroprotective roles
by suppressing excitotoxicity of NMDA receptors, promoting regeneration
of synapses and inhibiting cell apoptosis(Miranda et al., 2018; Ren \&
Dubner, 2007; Yamada \& Nabeshima, 2004). Bax is a pro-apoptotic protein
which accelerates cell loss including neurons, which lead to memory
disorders(Sun et al., 2021) wheread Bcl-2 is one of anti-apoptotic
proteins antagonizing cell apoptosis. Neuroinflammation reduced
expression of BDNF and p-TrkB proteins, further disrupting the balance
of Bax and Bcl-2 generating cell apoptosis in the mPFC, hippocampus and
EC regions. ERK1/2 and CREB play a vital role in learning and memory in
the brain which are involved in regulating transcription factors and
promoting protein synthesis(Cao et al., 2013; Zheng et al., 2020). In
this study, the expression of p-ERK1/2 and p-CREB was decreased in the
mPFC and hippocampus of LPS mice, while in the EC, only the level of
p-CREB was downregulated and no difference was found in the expression
of p-ERK1/2. In addition, CaMK2 is essential for the learning process
and synaptic plasticity, and GluR1 is an important component of the
postsynaptic density and controls dendrite growth(Sanderson et al.,
2008; Vigil \& Giese, 2018). We found that neuroinflammation reduced the
expression of p-CaMK2 and p-GluR1 both in the mPFC and hippocampus,
while in the EC, only the level of p-CaMK2 was decreased. These
differences between the EC with the hippocampus and mPFC may be related
to the mutual regulation of neural circuits.

Previous studies have reported that multiple brain regions are involved
in the process of learning and memory, such as the mPFC, hippocampus and
EC(Tanimizu et al., 2017)\textsuperscript{,}(Opitz, 2014). Researchers
have demonstrated that the mPFC is the most vulnerable among them, and
the hippocampus is moderate, whereas the EC is relatively less
susceptible to neuroinflammation(Maiti, Muthuraju, Ilavazhagan, \&
Singh, 2008). But the volume change in the EC is used as the earliest
indicator to evaluate preclinical cognitive deficits leading to the
development of dementia(Rodrigue \& Raz, 2004). The hippocampus is
responsible for storing information and is a key region involved in
learning and memory. The mPFC has been shown to play a vital role in the
process of attention, behavioral flexibility, social and emotional
behaviors, and its interactions with the hippocampus are involved in the
regulation of learning and memory(Euston, Gruber, \& McNaughton, 2012).
The EC is also related to spatial and long-term memory(Garcia \&
Buffalo, 2020). Neural projections from the mPFC or EC to the
hippocampus are known to be involved in the modulation of learning and
memory processes(Chao, de Souza Silva, Yang, \& Huston, 2020; Ladurelle
et al., 2011; Vertes, 2015). Lu et al. found that the
ECII\textsubscript{PN}-CA1\textsubscript{PV}pathway was impaired with
spatial learning and memory deficits in Alzheimer's disease mice, and
optogenetic activation of ECII\textsubscript{PN} rescued
ECII\textsubscript{PN}-CA1\textsubscript{PV} pathway defects and
alleviated the impairment of spatial learning and memory(X. Yang et al.,
2018). In addition, electrical stimulation of the EC alleviated spatial
memory deficits in Alzheimer's disease mice infusing amyloid peptides
1-42 into the hippocampus, which suggestes that there is functional
projection between the EC and hippocampus. However, it is not clear
whether the mutual projection of the mPFC, hippocampus and EC regions
and neuroregulation are associated with the metabolic changes in the
BDNF-TrkB signaling pathway and its downstream cascade.

There are still many limitations in this study. On the one hand, we only
tested protein levels of the BDNF-TrkB signaling pathway and its
downstream cascade in different brain regions, and no synaptic related
proteins were detected which directly indicates synaptic damages. On the
other hand, whether the changes in the BDNF-TrkB signaling pathway and
its downstream cascades among the mPFC, hippocampus and EC regions were
regulated by the projection of nerve fibers between these regions have
not been confirmed and deserves further investigation.

In summary, our research revealed that the BDNF-TrkB signaling pathway
and its downstream cascade disorders participated in learning and memory
impairments induced by neuroinflammation in mice (Figure 9).
Intraperitoneal injection of the TrkB agonist 7,8-DHF could effectively
alleviate cognitive dysfunction in LPS mice. As a result, the BDNF-TrkB
signaling pathway and its downstream cascade disorders are a new
viewpoint for learning and memory impairments induced by
neuroinflammation, and 7,8-DHF might serve as a potential target for
preventing or treating cognitive dysfunction induced by
neuroinflammation in neurodegenerative diseases.

\textbf{Author contributions}

Wen Zhang conceived the research, carried out the model building,
performed the Western blot, coordinated the lab work and drafted the
manuscript. Meng-meng Ge and Long-qing Zhang performed the statistical
analysis and drafted the manuscript. Xiao-man Yuan and Si-yi Han took
care of the NORT and drafted the manuscript. Anne Manyande, Xue-Bi Tian
and Yu-Ke Tian participated in its design and coordination and helped to
draft the manuscript. All authors read and approved the final
manuscript.

\textbf{Competing interest statement}

The authors declare no competing interests.

\textbf{Declaration of transparency and scientific rigor}

This paper adheres to the principles of transparent reporting and
scientific rigor in preclinical research stated in the BJP guidelines
for Design \& Analysis, Western blot, and Animal Experimentation and
recommended by funding agencies, publishers, and other organizations
committed to supporting this work.

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\textbf{Figure Legends}

\textbf{Abstract graphic.}

The BDNF-TrkB signaling pathway and its downstream cascades are involved
in the synthesis of synapses which then regulates learning and memory.
Intraperitoneal injection of LPS damages the BDNF-TrkB signaling pathway
and its downstream cascade leading to cognitive dysfunction. 7,8-DHF can
penetrate the blood-brain barrier and activate TrkB receptors,
alleviating learning and memory deficits.

\textbf{Figure 1. Behavioral tests and pro-inflammation levels in LPS
mice.} (A-C) There was no significant difference in the time spent in
exploring the same objects between the Control and LPS mice during the
training phase. In the testing phase, when exposed to the novel object,
LPS mice spent less time on the novel object and presented lower
discrimination ratio compared with the Control group (n=10). (D)
Compared to the Control mice, the concentration of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6 and TNF-\selectlanguage{greek}α
\selectlanguage{english}serum levels were significantly increased in LPS mice (n=4). Data are
presented as mean \selectlanguage{ngerman}± SEM. *\emph{P} \textless{} 0.05;
**\emph{P}\textless{} 0.01; ****\emph{P} \textless{} 0.0001.

\textbf{Figure 2. The protein levels of the BDNF-TrkB signaling pathway
and its downstream} \textbf{cascades in the hippocampus.} (A-D) Compared
with the Control mice, the levels of BDNF and p-TrkB proteins were
decreased in LPS mice, no difference was found in the expression of TrkB
(n=4). (E-G) The protein level of Bax was increased and the level of
Bcl-2 was reduced in the LPS group compared to the Control group (n=4).
(H-S) The protein expression of p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 in
LPS mice was lower than in Control mice. There was no statistical
difference in the levels of ERK1/2, CaMK2, CREB and GluR1 proteins
between the Control and LPS groups (n=4). Data are presented as mean ±
SEM. *\emph{P} \textless{} 0.05; **\emph{P} \textless{} 0.01;
***\emph{P} \textless{} 0.001.

\textbf{Figure 3. The protein levels of the BDNF-TrkB signaling pathway
and its downstream cascades in the mPFC.} (A-D) The protein levels of
BDNF and p-TrkB were reduced in LPS mice compared to the Control mice,
and no difference was found in the level of TrkB between these two
groups (n=4). (E-G) Compared with the Control group, the expression of
Bax was increased and the level of Bcl-2 was decreased in LPS group
(n=4). (H-S) The expression of p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 in
LPS mice were downregulated. There was no statistical difference in the
levels of ERK1/2, CaMK2, CREB and GluR1 proteins between the Control and
LPS groups (n=4). Data are presented as mean ± SEM. *\emph{P}\textless{}
0.05; **\emph{P} \textless{} 0.01; ***\emph{P} \textless{} 0.001;
****\emph{P} \textless{} 0.0001.

\textbf{Figure 4. The protein levels of the BDNF-TrkB signaling pathway
and its downstream cascades in the EC.} (A-D) The expression of BDNF and
p-TrkB were decreased in LPS mice compared to Control mice, and no
statistical difference was observed in the expression of TrkB between
these two groups (n=4). (E-G) The level of Bax was up-regulated and the
level of Bcl-2 was decreased in the LPS group compared to Control mice
(n=4). (H-S) The expression of p-CaMK2 and p-CREB was decreased in LPS
mice. There was no significant difference in protein levels of ERK1/2,
p-ERK1/2, CaMK2, CREB, GluR1 and p-GluR1 between the Control and LPS
groups (n=4). Data are presented as mean ± SEM. *\emph{P} \textless{}
0.05; **\emph{P} \textless{} 0.01.

\textbf{Figure 5. 7,8-DHF alleviated LPS-induced learning and memory
deficits in the mice.} (A-C) During the training phase, there was no
statistical difference in the time spent with objects after using
7,8-DHF or ANA12 in LPS mice. In the testing phase, the time spent on
the novel object and the discrimination ratio were increased in LPS mice
after administration of 7,8-DHF, however, ANA12 completely reversed the
effects of 7,8-DHF (n=10). Data are presented as mean ± SEM.
***\emph{P}\textless{} 0.001; ****\emph{P} \textless{} 0.0001; N.S.
\emph{P}\textgreater{} 0.05.

\textbf{Figure 6. 7,8-DHF restored the LPS-induced BDNF-TrkB signaling
pathway and its downstream cascade disorders in the hippocampus.} (A-D)
There were no statistical changes in the expression of BDNF and TrkB in
LPS mice after using 7,8-DHF or ANA12. The expression of p-TrkB was
increased in LPS mice after using 7,8-DHF, however, ANA12 reversed the
effects of 7,8-DHF (n=5). (E-G) 7,8-DHF decreased the expression of Bax
and increased the level of Bcl-2 in LPS mice, while ANA12 completely
reversed these changes (n=5). (H-S) The expression of p-ERK1/2, p-CaMK2,
p-CREB and p-GluR1 in LPS mice was increased after using 7,8-DHF,
however, ANA12 reversed the effects of 7,8-DHF. No statistical
differences were observed in protein levels of ERK1/2, CaMK2, CREB and
GluR1 in LPS groups after using 7,8-DHF or ANA12 (n=5). Data are
presented as mean ± SEM. *\emph{P} \textless{} 0.05;
**\emph{P}\textless{} 0.01; ***\emph{P} \textless{} 0.001; N.S.
\emph{P}\textgreater{} 0.05.

\textbf{Figure 7. 7,8-DHF restored the LPS-induced BDNF-TrkB signaling
pathway and its downstream cascades disorder in the mPFC.} (A-D) There
were no significant differences in protein levels of BDNF and TrkB in
LPS mice after administration of 7,8-DHF or ANA12. The level of p-TrkB
was increased in LPS mice after administration of 7,8-DHF, however,
ANA12 reversed the effects of 7,8-DHF (n=5). (E-G) The level of Bax was
reduced and the level of Bcl-2 was increased in LPS mice after
administration of 7,8-DHF, while ANA12 completely reversed these changes
(n=5). (H-S) The levels of p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 in LPS
mice were increased after administration of 7,8-DHF, however, ANA12
reversed the effects of 7,8-DHF. There were no statistical differences
in protein levels of ERK1/2, CaMK2, CREB and GluR1 in LPS groups after
administration of 7,8-DHF or ANA12 (n=5). Data are presented as mean ±
SEM. *\emph{P} \textless{} 0.05; **\emph{P} \textless{} 0.01;
***\emph{P} \textless{} 0.001; N.S. \emph{P} \textgreater{} 00.5.

\textbf{Figure 8. 7,8-DHF restored the LPS-induced BDNF-TrkB signaling
pathway and its downstream cascade disorder in the EC.} (A-D) No
statistical difference was observed in the expression of BDNF and TrkB
in LPS mice after using 7,8-DHF or ANA12. 7,8-DHF clearly increased the
expression of p-TrkB in LPS mice, while ANA12 reversed the therapeutic
effects of 7,8-DHF (n=5). (E-G) 7,8-DHF reduced the level of Bax and
increased the level of Bcl-2 in the LPS group, however, ANA12 reversed
these changes (n=5). (H-S) The expression of p-CaMK2 and p-CREB was
increased in LPS mice after administration of 7.8-HDF, while ANA12
completely reversed the effects of 7,8-DHF. There was no significant
difference in protein levels of ERK1/2, p-ERK1/2, CaMK2, CREB, GluR1 and
p-GluR1 in LPS groups after using 7,8-DHF or ANA12 (n=5). Data are
presented as mean ± SEM. *\emph{P} \textless{} 0.05;
**\emph{P}\textless{} 0.01; ***\emph{P} \textless{} 0.001; N.S.
\emph{P}\textgreater{} 00.5.\selectlanguage{english}
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