AMI, an Indazole Derivative, Improves Parkinson’s Disease by Inhibiting Tau Phosphorylation

Article abstract of DOI:10.3389/fnmol.2020.00165

Dopaminergic neuronal loss is the main pathological character of Parkinson’s disease (PD). Abnormal tau hyperphosphorylation will lead to dopaminergic neuronal loss. An indazole derivative 6-amino-1-methyl-indazole (AMI) successfully synthesized to inhibit tau hyperphosphorylation may exert a neuroprotective effect. The in vitro study showed that AMI effectively increased cell viability and alleviated the apoptosis induced by MPP⁺ in SH-SY5Y cells. In addition, AMI treatment significantly decreased the expression of p-tau and upstream kinases GSK-3β. In the MPTP-induced PD mice models, we found AMI apparently preserved dopaminergic neurons in the substantia nigra and improved the PD behavioral symptoms. Our results demonstrate that AMI exerts a neuroprotective effect by inhibiting tau hyperphosphorylation, representing a promising new candidate for PD treatment.

Full text of DOI:10.3389/fnmol.2020.00165

ORIGINAL RESEARCH
published: 19 November 2020
doi: 10.3389/fnmol.2020.00165
AMI, an Indazole Derivative,
Improves Parkinson’s Disease by
Inhibiting Tau Phosphorylation
Zhang Mao1†, Zhu Wen-ting2†, Wang Hai-tao1, Yu Hui3, Lan Shi-yi1, Xu Jiang-ping1* and
Wang Wen-ya1*
1School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China, 2The Third Affiliated Hospital of
Guangzhou Medical University, Guangzhou, China, 3School of Basic Medical Science, Southern Medical University,
Guangzhou, China.
Dopaminergic neuronal loss is the main pathological character of Parkinson’s disease
(PD). Abnormal tau hyperphosphorylation will lead to dopaminergic neuronal loss. An
indazole derivative 6-amino-1-methyl-indazole (AMI) successfully synthesized to inhibit
tau hyperphosphorylation may exert a neuroprotective effect. The in vitro study showed
that AMI effectively increased cell viability and alleviated the apoptosis induced by MPP+
in SH-SY5Y cells. In addition, AMI treatment significantly decreased the expression of
p-tau and upstream kinases GSK-3β. In the MPTP-induced PD mice models, we found
AMI apparently preserved dopaminergic neurons in the substantia nigra and improved
the PD behavioral symptoms. Our results demonstrate that AMI exerts a neuroprotective
effect by inhibiting tau hyperphosphorylation, representing a promising new candidate
for PD treatment.
Edited by:
Isabel Lastres-Becker,
Autonomous University of Madrid,
Spain
Reviewed by:
Begoña Villar-Cheda,
University of Santiago de
Compostela, Spain
Keywords: 6-amino-1-methyl-indazole, Parkinson’s disease, tau, SH-SY5Y, MPTP
Ana Isabel Rodríguez Pérez,
University of Santiago de
Compostela, Spain
INTRODUCTION
*Correspondence:
Xu Jiang-ping
jpx@smu.edu.cn
Wang Wen-ya
Parkinson’s disease (PD) is a common neurodegenerative disease. The apoptosis of substantia nigra
dopaminergic neurons plays a pivotal role in the pathogenesis of PD (Del Rey et al., 2018). The Lewy
bodies or neurites (Power et al., 2017; Liu et al., 2019a) was found in the advanced PD. Therefore,
we investigate the signaling pathways that regulate neuronal apoptosis as a novel therapeutic target
of PD.
wangwy@smu.edu.cn
†These authors have contributed
equally to this work
Tau protein is an important microtubule-associated protein in the central nervous system, which
mainly regulate by phosphorylation. Its induces and promotes the aggregation of microtubules
(Lee et al., 2019). Hyperphosphorylated tau interacts with α-synuclein to promote aggregation and
fibrosis, causing the formation of Lewy bodies and dysfunction of axonal transport (Singh et al.,
2019). Moreover, hyperphosphorylation and aggregation result in the formation of paired helical
filaments (PHFs; Zhou et al., 2018). Taltirelin, a long-acting TRH analog, down-regulated the levels
of p-tau (S396), exhibiting the neuroprotective effect in both cellular and animal models of PD
(Zheng et al., 2018). Formulated Chinese Medicine Shaoyao Gancao Tang reduces tau aggregation
and exerts neuroprotection (Chen et al., 2018). Together, phosphorylation of tau protein mediates
the pathological process of Alzheimer’s disease (AD) or PD, which is a potential therapeutic target
treatment for neurodegenerative disease (Winer et al., 2018; Chen et al., 2019).
Received: 28 January 2020
Accepted: 11 August 2020
Published: 19 November 2020
Citation:
Mao Z, Wen-ting Z, Hai-tao W, Hui Y,
Shi-yi L, Jiang-ping X and Wen-ya W
(2020) AMI, an Indazole Derivative,
Improves Parkinson’s Disease by
Inhibiting Tau Phosphorylation.
Front. Mol. Neurosci. 13:165.
doi: 10.3389/fnmol.2020.00165
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Mao et al.
AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
Indazole derivatives have multi-pharmacological activities
1-methyl-indazole. Subsequently, 6-nitro-1-methyl-indazole was
dissolved in methanol, palladium on carbon was added as a
catalyst, the reaction of hydrogen through 4 h, and finally filtered
and separated by column chromatography to obtain AMI.
such as anti-inflammatory, antibacterial and anti-tumor activities
(Denya et al., 2018; Liu et al., 2019b). Our previous studies have
found that 6-hydroxy-1H-indazole, 5-hydroxy-1H-indazole and
6-nitro-1H-indazole inhibit the phosphorylation of tau protein,
and have protective effects on MPP+-induced apoptosis of
SH-SY5Y cells. Particularly, 6-hydroxy-1H-indazole has shown MTT
a neuroprotective effect on 100 µm MPP+-induced apoptosis
of SH-SY5Y cells (Liang et al., 2016). In this study, 6-nitro-1H-
indazole was used as the mother nucleus to further optimize the
drug structure, and to explore the indazole derivatives, which can
specifically inhibit the phosphorylation of tau protein. Since the
drug needs to enter the brain to treat PD, our group introduced
a 1-position of methyl to 6-nitro-1H-indazole for increasing
the liposoluble of the compound, and finally the novel small
molecule 6-amino-1-methyl-indazole (AMI) was obtained.
In this study, we examined the effect of AMI in a PD model.
MTT assays and Hoechst 33258 staining were used to assess the
protective effect of AMI on SH-SY5Y cells after MPP+ treatment.
Meanwhile, MPTP-induced PD animal models were used to
detect the effects of AMI in vivo. The levodopa (L-DOPA) is the
first-line treatment of PD, we chose L-DOPA as a control drug in
our experiments.
MTT assay was used to determine the cell viability for SH-SY5Y
cells. The SH-SY5Y cells were seeded at a density of 1 × 104
cells/well in 96-well plates and incubated overnight. Firstly,
different concentrations of MPP+ acts on SH-SY5Y cells, and
confirm the optimal concentration of MPP+ on the cells. The
addition of MPP+ to SH-SY5Y cultures was performed at
300 µM. Afterwards, the cells were pretreated with different
concentrations of AMI for 2 h, then added with MPP+ at a final
concentration of 300 µM for 48 h. Add 100 µl of 0.5 mg/ml MTT
working solution to each well, and incubate for 4 h at 5% CO2 at
37◦C. The supernatant was discarded, each well was added 100 µl
DMSO, and shake at low speed for 10 min to fully dissolve the
crystals. The absorbance of each group of samples was measured
with a microplate reader (Bio-Rad Model 680) at a wavelength of
570 nm. Untreated cells represented 100% viability.
Hoechst 33258 Staining
MATERIALS AND METHODS
Material and Antibody
The SH-SY5Y cell line was pretreated with AMI for 2 h, and then
MPP+-induced apoptosis for 48 h. The cells were fixed 10 min
with fixative. After the fixative was removed, it was washed
twice with PBS for 3 min each time and then 0.5 ml of Hoechst
33258 staining solution was added, staining the cell line for 5 min.
After the staining solution was removed, the culture solution was
washed twice with PBS for 3 min. A drop of anti-fluorescence
quenching sealant was applied to the slide. The slide was covered
with a cell-coated cover glass, and blue nuclei were detected by
a fluorescence microscope. The excitation wavelength was about
350 nm and the emission wavelength was about 460 nm.
Methyl-4-phenylpyridine
(MPP+;
#D048),
1-methyl-4-
phenyl-1,2,3,6-tetrahydro-pyridine (MPTP; #M0896) and
levodopa (#D9628) were obtained from Sigma (St. Louis,
MO, USA). An ABC reagent Box (Vector PK-6101 Rabbit
IgG) and Golgi staining kit (PK401) were obtained from FD
NeuroTechnologes (Columbia, MD, USA). P-tau (Ser396;
#ab109390) and t-tau (#ab32057) were purchased from
Abcam (Cambridge, MA), while GSK-3β (#12456) and
phosphorylated GSK-3β (p-GSK-3β, ser9, #9323) were
purchased from Cell Signaling Technology (Danvers, MA,
USA). Anti-tyrosine hydroxylase (TH) was obtained from
Santa Cruz (Dallas, TX, USA). Immoblilon PVDF membranes
(#ISEQ00010) and Immobilon Western Chemiluminescent
HRP Substrate (#WBKLS0100) were purchased from Merck Co.
(Darmstadt, Germany).
SPF grade C57BL/6 mice, male 6–7 weeks, weight 22–27 g,
were purchased from Guangdong Experimental Animal Center,
license number: SYXK (Yue) 2016–0167, then free drinking
water, feeding to 10–11 weeks. All of the experimental
protocols were approved by the Institutional Animal Care and
Use Committee of Southern Medical University (Guangzhou,
Guangdong, China).
Western Blot Analysis
Western blott was used to determine the expression of GSK-
3β, p-GSK-3β(ser9), Tau, p-Tau(ser396) and TH. After drug
treatment, cells or tissues were quickly collected and lysed
with RIPA buffer containing 1% protease and 1% phosphatase
inhibitor, and shaken on ice for 40 min. The lysate was then
centrifuged at 12,000 rpm for 20 min at 4◦C and 10 min at
12,000 rpm. The supernatant was collected and the protein
concentration was determined using a BCA protein assay kit. An
equal amount of protein was separated by SDS-polyacrylamide
gel electrophoresis and transferred to a 0.4 µm pore size
hydrophilic PVDF membrane. Non-specific binding was blocked
with 5% w/v BSA, and membranes were then incubated in
milk dissolved in TBST for 2 h at room temperature. The
membrane was washed three times with TBST for 10 min,
and then incubated with primary antibodies at 4◦C overnight.
After rinsing and incubation with secondary antibodies, the blot
signals were detected with a Bio-Rad Ultraviolet Imaging System,
and immunoblotting was quantified with ImageJ software.
Synthesis of AMI
6-nitro-1H-indazole was dissolved in dimethylformamide and
then methyl iodide and sodium cyanide were added according
to the molar ratio of 6-nitro-1H-indazole: iodomethane: sodium
hydride = 1:2:2. The reaction was stirred for 24 h, then
filtered and extracted three times with ethyl acetate, dried over
anhydrous sodium sulfate, and evaporated to produce 6-nitro-
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AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
solution consisting of solution D, solution E and double
distilled water = 1:1:2. Neuronal morphology was observed
under a microscope.
Parkinson Animal Model Preparation and
Administration
The C57BL/6 male mice were divided into five groups (12 in
each group). The control group received normal saline. The
PD model group was treated with 30 mg/kg MPTP(dissolved
in normal saline) by intraperitoneal (i.p.) injection daily for
5 consecutive days (Schildknecht et al., 2017). Mice were treated
with AMI (2 and 4 mg/kg/day, dissolved in normal saline
containing 3% DMSO) half an hour before MPTP treatment.
The L-DOPA group was given 10 mg/kg/day (dissolved in
normal saline containing 3% DMSO + 0.5% CMC-Na, i.p.), 2 h
after MPTP treatment. Meanwhile, we recorded the incubation
period and duration of the mice after administration. The latent
period began with intraperitoneal injections of MPTP into mice
until paralysis occurred. Ten days after the final injection of
MPTP, the mice conducted behavioral experiments and then
sacrificed (Figure 3A).
For the analysis of dendritic branches, non-axonal synapses
longer than 10 µm and directly from the cell body are
defined as primary dendrites. All protrusions that emerge
from the primary tree are called secondary dendrites, and all
middle-end protrusions below 10 µm are called axonal tips.
When performing a sholl analysis, multiple concentric circles
centered on the cell body that differ in diameter by 15 µm
from each other, and all protrusions in the concentric circles
are counted.
Behavioral Experiment
Pole-climbin, rotarod test and traction test were performed
10 days after the final injection of MPTP (Wang et al., 2007).
There are 6–8 mice per group, all behavioral tests were conducted
in a double-blinded manner.
In traction test, the traction test measures muscle strength. A
cord with a diameter of 0.5 cm was placed 70 cm horizontally.
Each mouse’s front paws were hung on the rope and the mouse
was released. The time of the fall was recorded, as was whether
the mouse pulled its hind limbs to the rope. The feces and urine
of the mice were washed off before the experiment.
In rotarod test, a rotarod test is a common method to assess
neuromuscular coordination. First, mice were positioned on a
rotating rod (6 cm diameter) for 30 s, and then trained at a
constant speed of 12 rpm for 180 s. Sixty minutes after the last
training, a mouse was placed on the rod and the incubation
period of its fall was recorded as the end point measurement. The
average time of three trials was calculated for statistical analysis.
In pole-climbing test, Mice were placed near the top of rough
surface wooden stick (10 mm in diameter and 55 cm in height)
and their heads facing up. The total time for each animal to reach
the floor was recorded, with a maximum duration of 120 s. Each
mouse was tested three times at 10 min intervals and the average
time was calculated for statistical analysis.
TH Immunohistochemistry
Seven days after the last injection of MPTP, the ventral
midbrain was isolated and the TH content was detected
by immunohistochemistry. Brain slices (35 µm) were rinsed
with 0.3% Triton for 30 min, and then treated with freshly
prepared 3% H2O2 for 1 h. The brain slices were rinsed
twice in PBS for 10 min each, and rinsed for 1 h with
serum, then incubated overnight with rabbit anti-TH (1:10,000,
3%BSA with PSB) at 4◦C. Brain slices were then washed
three times with PBS for 10 min at room temperature and
incubated with the secondary antibody of the kit for 1 h
at room temperature. Next, brain slices were incubated for
1 h in ABC kit (Vector PK-6101 Rabbit IgG) and washed
three times with PBS. Finally, the color was developed in
DAB for 2–5 min, the brain slices were rinsed with PBS, and
the slices were dried. After the patches were dehydrated and
transparent, they were photographed under a light microscope
at a magnification of ×400. The numbers of TH-positive
dopaminergic neurons in the substantia nigra of each mouse
were counted. The mean number of right and left sides
was regarded as the neuronal cell number of each mouse
(Wang et al., 2007).
Statistical Analysis
Statistical analyses were performed using SPSS 13.0. Results were
expressed as mean ± SD. One-way ANOVA was employed for
multiple group comparisons. The statistical significance was set
at P < 0.05.
Golgi Staining
To study the morphological changes of nerve dendrites and
dendritic spines in animal brains after drug treatment by
Golgi staining (Zhong et al., 2019). Seven days after the last
injection of MPTP, the substantia nigra slices were obtained
for Golgi staining. According to the Golgi staining (FD
Neuro Technologes) manufactures’manual, we mixed liquid
A and liquid B for 24 h and stored it in the dark. The
whole brain placed into the prepared staining solution in
the dark for 3 weeks at normal temperature, and transferred
to solution C kept in the dark for at least 72 h. Then
RESULT
Characterization of AMI
AMI was obtained by a two-step reaction (Figure 1A), and the
hydrogen spectrum (Figure 1B) and mass spectrum (Figure 1C)
shown a characteristic absorption peak as the AMI.
AMI Improves Apoptosis in SH-SY5Y Cells
midbrain was fixed with an embedding solution, sliced on a After MPP+ Treatment
frozen slicer (100 µm), attached to a glass slide, immersed
in solution C, and dried for sectioning. The sections were
washed twice with double distilled water for 4 min each time.
The sections were then incubated for 10 min in a working
The in vitro cytotoxicity of MPP+ against human SH-SY5Y
cells was by using the MTT assay. As shown in the results of
Figure 2A, MPP+ can inhibit the growth of human SH-SY5Y cells
in a dose-dependent manner. We chose 300 µM MPP+ for cell
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AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
FIGURE 1 | (A) Synthetic route 6-amino-1-methyl-indazole (AMI). (B) AMI hydrogen spectrum. (C) AMI mass spectrum.
experiment. SH-SY5Y cells were pretreated with 10 and 100 µM
AMI for 2 h, and treated with 300 µM MPP+ for 48 h. The
MTT assay indicated that AMI attenuating the cytotoxicity of
MPP+ of SH-SY5Y cells (Figure 2B). In addition, MPP+ reduced
the number of TH-positive cells and caused the nuclear volume
to shrink. However, nuclear pyknosis was significantly reduced
with AMI pretreatment for 2 h (Figures 2C,D). These results
indicate that AMI have protective intervention in MPP+-induced
apoptosis of SH-SY5Y cells.
slow movement. However, the symptoms of the AMI 2.0 mg/kg
and 4.0 mg/kg groups were significantly alleviated, with only
mild tremors, vertical hair, arched back, and vertical tail. At
the same time, the latency from the injection of MPTP to
the above symptoms and the duration of PD symptoms was
observed and recorded. As shown in Table 1, the latency of
PD symptoms in the AMI 2.0 mg/kg and 4.0 mg/kg groups
was significantly longer than that in the MPTP group, and
the duration was shortened. Additionally, the duration of PD
symptoms in mice was obviously shortened, but the latency
was not changed after treating with levodopa compared to the
MPTP group.
AMI Inhibits MPP+-Induced Tau (Ser396)
Hyperphosphorylation in SH-SY5Y Cells
MPP+-induced tau hyperphosphorylation in SH-SY5Y cells, and
300 µM MPP+-treated SH-SY5Y cells for 8 h, showed that the
expression of p-tau (Ser396) was highest, and then gradually
decreased (Figures 2E,F). Therefore, we chose 8 h detect the
effect of AMI on the phosphorylation level of tau. As shown
in Figures 2I,J, The 10 µM and 100 µM AMI apparently
reduced the level of p-tau (Ser396) after MPP+ treatment, total
tau expression was not affected. Furthermore, MPP+ treatment
markedly inhibited the expression of p-GSK3β(Ser9). However,
pretreatment of AMI increased the phosphorylation of GSK3β
in SH-SY5Y cells (Figures 2G,H). In general, we found that
AMI reducing tau hyperphosphorylation may be related to its
inhibition of GSK-3β activity.
AMI Increases DA Neurons in C57/BL Mice
After MPTP Treatment
TH is an important and specific rate-limiting enzyme for DA
synthesis, which has a labeling effect on DA neurons. The
greater the number of TH-positive cells, the greater the content
of DA. We used immunohistochemistry and western blot to
detected TH expression in substantia nigra of the midbrain. As
shown in Figure 3B, the survival rate of neurons in the MPTP
group obviously decreased compared to the control group, which
proved that the PD animal model was successfully established.
However, the number of neurons in the mice pre-administered
with 2.0 mg/kg AMI (65.80 ± 12.8%, p < 0.001) and 4.0 mg/kg
AMI (64.13 ± 5.7%, p < 0.001) was higher than that in
the MPTP group (34.84 ± 2.7%; Figures 3B–F). Additionally,
the TH positive neurons in L-DOPA group (34.86 ± 4.9%)
was similar to the model group. These results indicated that
AMI rescues the loss of dopamine neurons in mice and
AMI Improves Symptom in C57/BL Mice
After MPTP Treatment
The symptoms of the mice were observed after administration
of MPTP and AMI. The MPTP group showed symptoms of
whole-body tremor, vertical hair, arched back, vertical tail, and
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AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
FIGURE 2 | AMI inhibits apoptosis of SH-SY5Y cells induced by MPP+. (A) The MTT assay was performed to assess the effect of MPP+ on the proliferation of
SH-SY5Y cells. (B) The MTT assay was performed to assess the effect of AMI on the proliferation of SH-SY5Y cells. Representative images (D) and quantitative data
(C) of Hoechst 33258 nuclear staining in SH-SY5Y cells, bar = 50 µM. (E) AMI increased intracellular p-GSK-3β (Ser9) levels in SH-SY5Y cells treated with MPP+.
(F) Densitometric quantification of p-GSK3β (ser-9)/t-GSK-3β protein levels. (G) MPP+ increased intracellular p-tau (Ser396) levels in SH-SY5Y cells.
(H) Densitometric quantification of p-tau (ser396)/t-tau protein levels in different time. (I) AMI decreased intracellular p-tau (ser396)/t-tau protein levels at 8 h.
(J) Densitometric quantification of p-tau(ser396)/t-tau protein levels at 8 h after treating with AMI. *p < 0.05, **p < 0.01, ***p < 0.001 vs. MPP+ group. #p < 0.05,
##p < 0.01 vs. vehicle group (control).
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AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
TABLE 1 | Effect of 6-amino-1-methyl-indazole (AMI) on the latency period and
duration of the MPTP model of PD symptoms in mice (n = 11–12, × ±s).
longer in each section of the rod, the scores of pole-climbing
test results show that the scores in 2.0 mg/kg AMI group
(8.0 ± 1.22), 4.0 mg/kg AMI group (7.60 ± 1.14), and L-DOPA
group (7.60 ± 0.55) were higher than those in MPTP group
(5.20 ± 1.64, p < 0.05; Figure 5C).
Group
Latency period (s)
Duration (s)
Control
MPTP
AMI (2 mg/kg)
AMI (4 mg/kg)
L-DOPA
∞
-
197.67 ± 22.91
225.77 ± 24.84∗
229.79 ± 22.04∗∗
200.27 ± 16.62
1039.84 ± 99.61
905.25 ± 58.96∗∗∗
899.75 ± 42.45∗∗∗
951.95 ± 53.23∗
Collectively, these results suggested that the mice in the
MPTP group had difficulty performing movements due to the
weakness of the hind limbs. The AMI 2.0 mg/kg and 4.0 mg/kg
groucomparisons. The statistical significanceps significantly
improved the obstacle, demonstrating smoother movements and
higher behavioral scores.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. MPTP.
has a neuroprotective effect, while levodopa has no neuronal
protective effect.
To further study whether AMI has a protective effect on DA
neurons, western blot analysis used to detect TH expression in
midbrain. The expression of TH protein in the MPTP group was
markerly lower than that in the control group. Compared with
the MPTP group, the 2 mg/kg and 4 mg/kg AMI intervention
groups increased the expression of TH protein. These results
further suggested that AMI can ameliorate the MPTP-induced
loss of TH in the substantia nigra striatum of mice, and
has a protective effect on the degeneration of DA neurons
(Figures 3C,D).
DISCUSSION
The most prominent pathological feature of PD is the loss
of dopaminergic neurons in the substantia nigra. Therefore,
developing a new drug that inhibits dopaminergic apoptosis
may significantly improve treatment effect of PD (Yang et al.,
2018a; Geibl et al., 2019). This study found that AMI has a
protective effect on MPP+-induced apoptosis of SH-SY5Y cells,
and this effect is accompanied by decreasing phosphorylation
level of tau.
The abnormal phosphorylation of tau protein is mainly due
to the imbalance of protein kinase and phosphatase regulation
(Lee et al., 2019; Singh et al., 2019). When the kinase activity
is increased and the phosphatase activity is decreased, the tau
protein is hyperphosphorylated. Protein kinases include GSK-3
and Cyclin-dependent Kinase 5 (CDK5), in which GSK-3β
phosphorylates multiple sites of the tau protein. Importantly,
GSK-3β is one of the upstream tau kinases, which involved
in the formation of tau phosphorylation and neurofibrillary
tangles in AD, and it is also the key to a variety of neuronal
survival signals to interfere with neuronal apoptosis (Yang et al.,
2018b; Chang et al., 2019; Shi et al., 2019). Recent studies have
identified GSK-3β as a potential therapeutic target in Alzheimer’s
disease (Shi et al., 2019). Cornel Iridoid Glycoside, a promising
agent for AD therapy, inhibits tau hyperphosphorylation via
regulating cross-talk between GSK-3β and PP2A signaling (Yang
et al., 2018a). Osthole decreases tau protein phosphorylation
via PI3K/AKT/GSK-3β signaling pathway in AD (Yao et al.,
2019). Consistent with previous studies, our results showed
that AMI inhibits the expression of the tau upstream kinase
GSK-3β. In addition, tau protein kinase II (TPKII) formed
by a complex containing two subunits of Cyclin-dependent
Kinase 5 (CDK5) and p35 can synergistically increase the
efficiency of GSK-3β phosphorylation of tau protein (Xiao
et al., 2018; Giannopoulos et al., 2019). Therefore, we need to
investigate the expression of tau protein kinase II (TPKII) in the
next experiment.
AMI Enhances Substantia Nigra Dendrites
in C57/BL Mice After MPTP Treatment
To explore the effect of AMI on the substantia nigra dendrites,
golgi staining was used to detect the density and length
of neuronal dendrites in the substantia nigra. As shown
in Figures 4A–E, the total number of dendritic branching
point in the substantia nigra of the midbrain were decreased
in MPTP group (3.75 ± 0.89, p < 0.01) compared with
the control group (9.62 ± 0.52). However, 4.0 mg/kg AMI
(8.25 ± 0.89, p < 0.01) significantly increased the total
number of dendritic branching point and dendritic length.
The 2.0 mg/kg AMI (4.25 ± 0.89) group may be due to
insufficient dose, demonstrating no obvious protective effect.
We also found that the in L-DOPA group (2.37 ± 0.74) has
a weak influence on the density of neuronal dendrites in the
substantia nigra of mice (Figures 4F,G). These results indicated
that AMI can effectively improve MPTP-induced dendritic spine
density reduction.
AMI Improves Behavioral Abnormalities in
C57/BL Mice After MPTP Treatment
We used traction test, rotarod test and pole-climbing test
to investigated the behavioral ability in different treat group.
As shown in Figure 5A, it was observed that the hind
paws of the MPTP group were weak and the paws could
not hold the wires, but the mice in other groups can hold
the wire with one or two paws. The scores showed that
2.0 mg/kg AMI group (2.50 ± 0.53), 4.0 mg/kg AMI group
(2.38 ± 0.52), L-DOPA group (2.71 ± 0.49) and control group
(2.86 ± 0.38) had no significant difference, however the scores
in MPTP group (1.40 ± 0.55, p < 0.001) was lower than
the control group. In the rotarod test, AMI and L-DOPA can
improve the MPTP induced postural agility and slow response
(Figure 5B). Finally, the mice in the MPTP group stayed
This study also suggested that AMI has a therapeutic
effect on the PD animal model in vivo. Continuous injection
high-dose MPTP for 5 days was used to prepare a PD
mice model, and levodopa was used as a positive control.
The dopamine neurons were destroyed by MPTP, and
dopamine levels were decreased in mice. Dopamine was
not replenished in a short time, so the behavioral abnormalities
of MPTP group became serious, but AMI protected the
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AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
FIGURE 3 | (A) Experimental dosing regimen. (B) Representative images and quantitative data of TH staining in the substantia nigra of midbrain. Magnification: 40×
(a, Control; b, MPTP; c, AMI 2 mg/kg; d, AMI 4 mg/kg; e, L-DOPA 10 mg/kg; f, the number of TH immunopositive cells in the substantia nigra). (C) TH protein
expression was examined by western blot analysis. (D) The protein levels of TH were quantified by densitometry. Slice thickness: 35 µm. ##p < 0.01 vs. control;
∗p < 0.01, ∗∗p < 0.001 vs. MPTP.
dopamine neurons of mice from MPTP injury, while L-DOPA
was supplemented with dopamine, which also improved
Parkinson’s behavioral abnormalities (Thomas et al., 2019).
On the contrary, the TH immunohistochemistry results
shown that number of DA neurons in the L-DOPA group
was not significantly higher than that in the MPTP group,
which further illustrates the current major drawbacks of
L-DOPA treatment with PD, these drugs can only improve
symptoms, but cann’t effectively delay the progression of
the disease and prevent the apoptosis of dopamine neurons.
In addition, with the long-term application of L-DOPA,
its efficacy gradually decreased, most patients will tolerate
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Mao et al.
AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
FIGURE 4 | Golgi staining for detecting the density and length of neuronal dendrites in the substantia nigra of the brain (A) Control; (B) MPTP; (C) AMI 2 mg/kg; (D)
AMI 4 mg/kg; (E) L-DOPA 10 mg/kg. (F) Total number of dendritic branching points. (G) Dendritic length. Magnification: 40×, slice thickness: 100 µm.
##p < 0.01 vs. control; ∗∗p < 0.001 vs. MPTP.
FIGURE 5 | AMI improved behavioral disorders in MPTP-induced PD model mice. (A) Rotarod test. (B) Traction test. (C) The Pole-climbing test. ##p < 0.01 vs.
control; ∗p < 0.01, ∗∗p < 0.001 vs. MPTP, n = 6–8.
complications, such as, dyskinesia or mental disorders DATA AVAILABILITY STATEMENT
(Wichmann, 2019).
The datasets generated for this study are available on request to
the corresponding author.
The maintenance of the density of striatum dendritic spines
can delay the exacerbation of PD, prolong the use time of
levodopa and improve its therapeutic effect (Deutch et al.,
2007). Golgi staining was used to observe the morphology of
neurons (Czechowska et al., 2019). AMI significantly increases
the density of neuronal dendrites in the substantia nigra of
mice. Notely, the increase of dendritic spine density of striatum
can compensate for PD animal striatum DA level reduction
and promote the recovery of their behavioral function (Kim
et al., 2013). Our behavioral experiment results are consistent
with literature reports. In general, AMI has neuroprotective and
neurotrophic effects. On the one hand, it protects the integrity
of dendritic spines, on the other hand, AMI saves the loss
of dopamine neurons, and effectively improves the behavioral
abnormalities of C57/BL mice.
ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional
Animal Care and Use Committee of Southern Medical University
(Guangzhou, Guangdong, China).
AUTHOR CONTRIBUTIONS
ZM conducted the experimental work, analysis and
interpretation of the data. ZW-t conceived and drafted the
Frontiers in Molecular Neuroscience | www.frontiersin.org
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November 2020 | Volume 13 | Article 165

Mao et al.
AMI Improves Parkinson’s Disease Related Pathologies by Inhibiting Tau Phosphorylation
manuscript. WW-y, XJ-p and WH-t designed the study
and critically revised the manuscript. Other authors were
involved in data collection and checked the manuscript.
All authors contributed to the article and approved the
submitted version.
FUNDING
This work was supported by the key project of the
Natural Science Foundation of Guangdong Province
(No. 2017A030313884).
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Mao, Wen-ting, Hai-tao, Hui, Shi-yi, Jiang-ping and Wen-ya.
This is an open-access article distributed under the terms of the Creative Commons
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