Ketamine Promotes Alzheimer’s-Like Neurodegeneration by Activating Glycogen Synthase Kinase 3 Beta and Inhibiting Protein Phosphatase 2A

*Corresponding Author:

Rui Fan

Department of Psychiatry, Tianjin Anding Hospital, Tianjin 300000, China

E-mail: [email protected]

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

Abstract

To investigate the mechanism of ketamine promoting Alzheimer’s-like neurodegeneration in mice. 24 male 8 w old Kunming mice of specific pathogen-free grade were randomly divided into a blank control group (control group) and a ketamine intervention group (experimental group) according to the random number table, 12 mice in each group. The mice in the experimental group were given ketamine injection at the dose of 30 mg/kg. Mice in the control group were given an equal volume of normal saline injection. The intervention was conducted once a day for consecutive 6 mo. The behavioral changes of the mice in the two groups were compared. Western blot was used to detect the expression levels of tau (phospho Thr231), tau (phospho S396), tau (phospho Ser404), glycogen synthase kinase 3 beta, glycogen synthase kinase 3 beta (phospho ser9) and protein phosphatase 2A proteins in the brain tissue of hippocampus and the ratio of phosphorylated tau protein to tau protein was calculated. The expressions of beta-amyloid protein and tau protein in the hippocampus were observed by immunohistochemistry. The results of the Morris water maze showed that the escape latency of mice in both groups was gradually decreased and the escape latency of mice in the control group was lower than that in the experimental group. Besides, the times of crossing target platform and the proportion of activity time in target quadrant of mice in the experimental group were lower than those in the control group. At the same time, compared with the control group, the expression levels of tau (phospho Thr231) and tau (phospho Ser404) were decreased in the experimental group, and the expressions of tau (phospho S396), glycogen synthase kinase 3 beta, glycogen synthase kinase 3 beta (phospho ser9) and protein phosphatase 2A proteins were increased. Compared with the control group, the expression of beta-amyloid protein in the experimental group was significantly reduced and the opalescence density of beta-amyloid protein was significantly lower than that in the control group (p<0.05). Long-term use of ketamine can lead to up-regulated expression of beta-amyloid protein and tau protein in mice hippocampus, which may induce hyperphosphorylation of tau protein at Thr231, S396 and S404 by activating glycogen synthase kinase 3 beta and inhibiting protein phosphatase 2A, causing cognitive function decline in mice.

Keywords

Ketamine, cognitive function, tau protein, glycogen synthase kinase 3 beta, beta-amyloid
protein, neurodegeneration

Ketamine is a kind of non-phenobarbital intravenous
anesthetic. Thanks to the advantages including low
respiratory inhibition, short duration, fast absorption and
quick anesthesia, it is widely used in clinical practice[1,2].
Due to its mental dependence and hallucinogenic
effect, ketamine has become a type of synthetic drug
abused in China and has been included in psychotropic
drugs. Studies have proven that ketamine can damage
the cognitive, executive, visual-spatial ability and other
cognitive functions of abusers through N-Methyl-DAspartate
(NMDA) receptors, monoamine receptors,
acetylcholine receptors, voltage-gated calcium channel
receptors, opioid receptors and so on[3,4]. Tau protein is a microtubule-associated protein mainly distributed
in axons and dendrites of neurons, which has the
functions of regulating the growth and development of
neurons, maintaining the morphology and stability of
microtubules[5]. Beta (β)-amyloid protein is formed by
cleavage of β-amyloid precursor protein by secretase,
which mainly resides in astrocytes and neurons. It is neurotoxic and causes cell damage and death by causing
hyperphosphorylation and prominent changes of tau
protein[6]. Studies have proven that the up-regulation
of tau protein and β-amyloid protein expression is
related to ketamine-induced cognitive impairment[7]. In
this study, a ketamine abuse model was established to
explore the mechanism of cognitive impairment caused
by abusing ketamine, so as to find a new target for
clinical treatment.

Materials and Methods

Experimental animals:

Twenty-four 8 w old male Kunming mice of Specific
Pathogen-Free (SPF) grade were purchased from
Beijing Vital River Laboratory Animal Technology
Co., Ltd, approval No: SCXK (J) 2016-0011. The mice
were fed adaptively in the animal room with a room
temperature of 22°±1° and a light/dark cycle of 12 h.
They were free to eat or drink.

Main experimental instruments and reagents:

Main experimental instruments: Upright fluorescent
microscope (Olympus, Japan), embedding machine
(Tianzhirui Medical Technology, China), slicing
machine (Leica, Germany), -80° refrigerator (Haier,
China), drying oven (Senxin Experimental Instrument
Co., Ltd., China), centrifuge (Xiangyi Laboratory
Instrument Development Co., Ltd., China).

Main experimental reagents: Preparation of 30 mg/ml
ketamine solution by ketamine hydrochloride injection
(Fujian Gutian Pharmaceutical Co., Ltd., batch No.:
H35020148, specification: 100 mg/2 ml) and normal
saline were blended by a ratio of 3:2 and then stored
in the refrigerator at 4°. Radioimmunoprecipitation
Assay (RIPA) protein lysate (Biyuntian Biotechnology,
China), protease inhibitor mixture (Millipore, USA),
Glycogen Synthase Kinase 3β (GSK-3β) antibody
(Merck, Germany) and Protein Phosphatase 2A (PP2A)
antibody (Merck, Germany).

Experimental methods:

Animal grouping and intervention methods: Using
the random number table, the mice were randomized
into two groups as blank control group (control group)
and ketamine intervention group (experimental group),
with 12 mice in each, they were intervened following 2
w of adaptive feeding.

The mice were fixed in the position of low tail and
high head to migrate their viscera and the needle was inserted at the depth of 5 mm from the white line of
their lower abdomen, the syringe was pushed forward
subcutaneously 2 cm at an angle of 45°. The feeling
of negative pressure attraction of the abdominal cavity
without foreign body suction indicates successful
insertion and then the injection can be performed. The
left and right abdomen was injected alternately. The
mice in the experimental group were injected with
ketamine at a dose of 30 mg/kg; those in the control
group were injected with the same volume of normal
saline. Following the injection, the mice in each group
were put into a separate cage to observe their skin color
to prevent hypoxemia. After the righting reaction was
restored in each group, the mice were replaced into
the cage. The above-mentioned interventions were
performed once a day for consecutive 6 mo.

Morris water maze: After 6 mo of intervention,
Morris water maze test was carried out on the mice. A
pool with a diameter of 1 m and a depth of 0.4 m was
selected and the water temperature was 20°±1°. Note
that the experimental environment should be quiet and
not affected by external factors. A target platform with
a diameter of 5 cm was placed in the target quadrant,
about 1 cm below the water surface. 1 d before the
formal experiment, each mouse in the two groups was
put into the water for swimming for 1 min in order to
adapt to the environment. The formal experiment lasted
for 6 d, the mice were trained from 1 to 5 d. Facing
the pool wall, they were put into in the pool from the
entry points in the 1^st to 4^th quadrants and the time
required for the mice to climb the platform in the target
quadrant, namely escape latency, was recorded. If the
mice did not climb onto the target platform within 60 s,
it was recorded as 60 s, and they was artificially guided
to the platform and stayed for 10 s. A place navigation
test was carried out on the 6^th d and the mice were put
into water from entry point at the opposite side of the
target quadrant, and the escape latency was recorded.
Then the platform was removed and the mice were put
into water from entry point at the opposite side of the
target quadrant. The frequency of crossing the platform
within 60 s and the residence time in the target quadrant
was recorded.

Brain tissue sampling: The mice were anesthetized
by intraperitoneal injection of chloral hydrate at the
dose of 300 mg/kg and then their limbs were fixed.
The chest was dissected to expose the heart, the needle
was inserted at the apex beat of the left ventricle, the
right atrial appendage was cut open and normal saline
and heparin were injected until the liver turned white.

Finally, they were perfused with 4 % paraformaldehyde
solution, the brain was taken out on the ice after the
tail and limbs were stiffened and then the head was
amputated. With reference to the stereotaxic atlas for
mouse brain and the hippocampal tissue was bluntly
separated along the coronal plane on the posterior side
of the pituitary gland.

If the Western blotting experiment was carried out,
the mice would be anesthetized, decapitated and the
brain tissue was taken out on the ice to separate the
hippocampal tissue. The stripped hippocampal tissue
was placed in a cryopreservation tube and frozen at
-80° with liquid nitrogen.

Western blotting test: The Phenylmethylsulfonyl
Fluoride (PMSF) protease inhibitor of 100 mmol/l
concentration was mixed with RIPA protein lysate at the
ratio of 1:100 and the hippocampal tissue was weighed.
The mixture obtained was added to hippocampal
tissue at the ratio of 100 μl lysate per 100 mg tissue
and the test tube was placed in ice-water mixture for
homogenization. After centrifuging for 15 min at 4°
and 12 000 rad/min, the supernatant was collected. The
protein concentration of each sample was determined
by Bicinchoninic Acid (BCA) assay method and the
lysate was used to balance the concentration. The 5x
loading buffer was added to the protein sample, at
the ratio of 1:4. It was denatured in boiling water at
100° for 10 min, then cooled to room temperature and
stored at -20°. After mixing the same amount of protein
samples from each group, the sample was injected
into the sodium dodecyl sulfate–polyacrylamide gel
electrophoresis system with a pipette and treated for 30
min at constant pressure of 85 V, then for 85 min at 115
V and the electrophoresis ended when bromophenol
blue ran to 1 cm at the bottom of the glass plate. Then
the membrane was transferred and was treated for 35
min with 25 V. Following the transfer, the target protein
area was cut in the Polyvinylidene Difluoride (PVDF)
membrane, then placed into the confining liquid after
washing off the transfer buffer and then oscillated gently
on the shaker. It was incubated overnight at 4° after the
addition of the primary antibody. After rewarming at
30° at room temperature, the PDVF membrane was
rinsed for 3 times, 10 min each and then placed in the
secondary antibody and incubated on the shaker for 1
h at room temperature, and then the PDVF membrane
was rinse again, 10 min each time, for a total of 3 times.
The PDVF membrane was placed in the gel imager; an
appropriate amount of chemiluminescence substrate
was dripped into on the surface of the membrane and then exposed later. The protein bands were scanned
and analyzed by Image J software to calculate the gray
level.

Immunohistochemical test: After the separation,
the hippocampus was put into 4 % paraformaldehyde
and fixed for 48 h at 4°. The tissue was placed in an
embedding cassette and washed with running water
for 30 min. After gradient dehydration with alcohol,
transparent treatment with xylene and paraffin
embedding were performed and then it was cooled and
solidified for standby. After the tissue was trimmed to
expose the observation surface, the slice thickness of the
slicer was set to 5 μm and continuous slices were made
along the coronal plane. After spreading the slices, they
were placed in the thermostat at 65° overnight. The
dewaxing and rehydration were performed, then an
appropriate amount of sodium citrate repair solution
was added to the antigen repair box and then the repair
box was heated to above 90° in a pressure cooker. The
slices were placed in the box, heated to boiling for 8
min, then cooled at room temperature, rinsed with
Phosphate Buffered Saline (PBS) for 5 min each time,
a total of 3 times. 3 % hydrogen peroxide was added to
the slices, incubated for 10 min at room temperature,
then rinsed with PBS for 5 min each time, a total of 3
times. 10 % goat serum was added to the slices and was
sealed for 30 min at room temperature. After removing
the confining solution, primary antibody was added
and sealed at 4° overnight. After rewarming 30 min at
room temperature, rinsed with PBS for 3 min each time,
a total of 3 times. An appropriate amount of polymer
enhancer was added into the slices, incubated at room
temperature for 30 min and rinsed for 5 min each time,
a total of 3 times. After removing the PBS buffer, an
appropriate amount of 3,3′-Diaminobenzidine (DAB)
substrate was added to the slices. The staining can be
terminated after obvious stain was observed under the
microscope. After gradient dehydration with alcohol,
the slices were treated transparently with xylene, sealed
with neutral sizing agent, then observed and preserved
after drying. The optical density of the sample photos
was measured semi-quantitatively by Imagine-Pro Plus
software.

Statistical method:

Statistical Package for the Social Sciences (SPSS) 20.0
was used for statistical analysis of the data, the counting
data was presented by the mean±standard deviation and
the t-test was performed. p<0.05 means the difference
is of statistical significance.

Results and Discussion

It was shown that the escape latency of the two groups
decreased gradually and that of the experimental group
was longer than that of the control group, suggesting
statistically significant difference (p<0.05) as shown in Table 1. The frequency of crossing the target platform
and the proportion of activity time in the target quadrant
in the experimental group were lower than those in
the control group, suggesting statistically significant
difference (p<0.05) as shown in Table 2.

Table 1: Comparison of Escape Latency Between Two Groups of Mice (x±s)

Table 2: Comparison for The Frequency of Crossing The Target Platform and The Proportion of Activity Time in The Target Quadrant Between The Two Groups (x±s)

The findings indicated that compared with the control
group, the expression of tau (phospho Thr231) and
tau (phospho Ser404) protein decreased, while the
expression of tau (phospho S396) protein, GSK-3β
protein, GSK-3β (phospho Ser9) protein and PP2A
protein increased in the experimental group, and the
difference was statistically significant (p<0.05) as
shown in Table 3.

Table 3: Results of Western Blotting Test in Two Groups of Mice (β-actin, x±s)

The results indicated that compared with the control
group, tau (phospho Thr231)/tau and tau (phospho
Ser404)/tau decreased, while tau (phospho S396)/tau
increased in the experimental group as shown in Table
4.

Table 4: Mouse In The Two Groups Result Ratio of TAU (PHOSPHO THR231) Protein, TAU (PHOSPHO S396) Protein, TAU (PHOSPHO SER404) Protein To TAU Protein (x±s)

The findings suggested that expression of yellow or
brown β-amyloid protein and tau protein were positive
in both groups. Compared with the control group, the
expression of β-amyloid protein decreased significantly
and that of tau protein increased significantly in the
experimental group (p<0.05). And compared with the
control group, the optical density of β-amyloid protein
was significantly lower and that of tau protein was
significantly higher in the experimental group (p<0.05)
as shown in Table 5.

Table 5: Comparison of Average Optical Density of Hippocampal Protein Between Two Groups Of Mice (x±s)

Ketamine, a commonly used anesthetic in clinical
practice, has become a recreational drug abuse
due to its hallucinogenic effect and psychological
dependence[8]. Studies have shown that ketamine
abuse has obvious toxic side effects on nervous system
and mental function, mainly manifested by cognitive
impairment, such as memory and cognitive decline,
slow response, decreased computing power and other
neurodegenerative changes[9].

Cognitive function is one of the most basic and
important functions of the nerve center, which is
regulated by many brain regions such as cerebral
cortex, hippocampus, corpus striatum and so on. When
the cognitive function is impaired, the related learning,
memory, thinking and other processes are abnormal,
such as memory impairment, usually with agnosia,
aphasia and other changes[10,11]. The hippocampus is
located in the deep layer of the brain tissue and belongs
to the limbic system. It is the center of information
processing, is closely related to memory, learning and
other cognitive functions and focuses on regulating
spatial memory and short-term memory[12]. Therefore,
the hippocampus was used as the target brain region
for this study and the mechanism of Alzheimer’s-like
neurodegenerative disease in mice was investigated by
establishing a ketamine abuse model.

Morris water maze is one of the commonly used
behavioral experimental methods, which evaluates the
abilities of mice such as learning, spatial positioning
and spatial memory by testing the parameters of their
movement to the target platform and quadrant[13].
The results showed that compared with the control
group, the escape latency of mice was higher and the frequency of crossing the target platform and the
proportion of activity time in the target quadrant were
lower, suggesting that ketamine abuse could damage
the cognitive function of mice and reduce their learning
ability and spatial ability.

Related studies have shown that ketamine-induced
cognitive decline in rats is associated with the
increased expression of β-amyloid protein and tau
protein and hyperphosphorylation of tau protein in the
hippocampus[14]. The injection of β-amyloid protein into
the hippocampus can also cause neuronal loss and tissue
necrosis at the injection site, accompanied by obvious
cognitive impairment. The hyperphosphorylation of tau
protein can lead to the decline[15] of cognitive function
by destroying microtubule homeostasis and axonal
transport function, inducing postsynaptic dysfunction
and blocking cell signal transduction. The ratio of
phosphorylated tau protein to tau protein can reflect the
phosphorylation level of tau protein in brain tissue[16].
Phosphatase and protein kinase are the primary
components that regulate the level of tau protein phosphorylation. GSK-3β and Cyclin-Dependent
Kinase 5 (CDK5) are the main protein kinases that
induce tau protein phosphorylation[17,18]. The former was
significantly induced at S396 and S404, while the latter’s
sites were S202, S235 and S404. The activity of GSK-
3β was negatively correlated with the phosphorylation
degree of enemy Ser9, which means the higher the
dephosphorylation degree of Ser9, the higher the level
of GSK-3β and the phosphorylation degree of related
tau protein will increase[19]. Tau protein phosphorylation
is also regulated by PP2A-based protein phosphatase,
which inhibits tau protein phosphorylation more
strongly than other protein kinases[20,21]. The results
indicated that the phosphorylation degree of tau protein
at the sites Thr231, S396 and S404 in the observation
group was higher than that in the control group,
which may be associated with the increase of GSK-3β
protein expression and the decrease of PP2A protein
expression[22-24].

In conclusion, long-term use of ketamine can lead to
up-regulated expression of β-amyloid protein and tau protein in the hippocampus of mice, which may induce
hyperphosphorylation of tau protein at Thr231, S396
and S404 by activating GSK-3β and inhibiting PP2A,
resulting in cognitive impairment in mice.

Conflict of interests:

The authors declared no conflicts of interest.

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