An Isoquinolinium Dual Inhibitor of Cholinesterases and Amyloid β Aggregation Mitigates Neuropathological Changes in a Triple-Transgenic Mouse Model of Alzheimer's Disease

Yaojun Ju, Harapriya Chakravarty, and Kin Yip Tam*

Research Article

An Isoquinolinium Dual Inhibitor of Cholinesterases and Amyloid β

Aggregation Mitigates Neuropathological Changes in a Triple-

Transgenic Mouse Model of Alzheimer’s Disease

Cite This: https://dx.doi.org/10.1021/acschemneuro.0c00464

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ABSTRACT: Alzheimer’s disease (AD) is a complex neurodegenerative

disorder aﬀecting millions of people worldwide. The underlying

pathologic mechanisms of AD are unclear. Over the decades, the

development of single target agent did not lead to any successful

treatment for AD. A multitarget agent that could tackle more than one

AD phenotype may be helpful as a treatment strategy. Cholinesterases

(

ChEs) including acetylcholinesterase (AChE) and butyrylcholinesterase

BuChE), are currently the drug targets with approved treatments.

Moreover, amyloid beta (Aβ) deposition is a hallmark of AD that

receives considerable attention. Herein, 9Q, a previously reported dual

target inhibitor dealing with cholinergic dysfunction and amyloid

deposition for AD treatment, has undergone thorough investigations.

In vitro studies revealed that 9Q exhibited over 80% inhibition of ChE

activity at 100 μM and more than 30% inhibition of Aβ aggregation at 1 mM concentration. Moreover 9Q was able to penetrate the

blood−brain barrier (BBB) and enhance the cerebral acetylcholine level in triple transgenic AD (3xTg-AD) mice. Following one

month treatment with 9Q, the amyloid burden and the cognitive deﬁcits in 3xTg-AD mice were signiﬁcantly ameliorated. It was

observed that 9Q treatment mitigated synapse dysfunction, decreased amyloidogenic APP processing, and reduced the tau pathology

in 3xTg-AD mice. Taken together, our results suggested that dual inhibition of cholinesterases and Aβ aggregation could be a

promising approach in AD treatment.

KEYWORDS: cholinesterase, amyloid beta, dual inhibition, Alzheimer’s disease

. INTRODUCTION

Cholinergic dysfunction was the ﬁrst hypothesis for AD

pathology. Donepezil, rivastigmine, galantamine, and mem-

antine are currently the only four AD drugs available in clinics.

The former three are acetylcholinesterase inhibitors (AChEI)

while the latter is an N-methyl-D-aspartate receptor (NMDAR)

antagonist, which could modulate the symptoms of AD to a

certain extent. It is noted that AChEIs (donepezil,

rivastigmine, and galantamine) account for most of the FDA

approved AD drugs. AChEIs decrease the acetylcholinesterase

Alzheimer’s disease (AD) accounts for most age-related

dementia cases with number predicted to reach 131.5 million

by 2050. It has brought heavy ﬁnancial burden to many

countries around the globe. Though considerable eﬀorts have

been made to discover therapeutic interventions, AD remains

inexorable and incurable. By now, only a few FDA approved

drugs are available to oﬀer limited cognitive improvement.

The underlying pathologic mechanisms of this disease are

unclear. Cholinergic dysfunction, amyloid plaques, neuro-

(

AChE) activity in AD patients’ brains, impede cerebral

ﬁbrillary tangles, neuroinﬂammation, and mitochondrial

dysfunction, as well as oxidative stress, are the major hallmarks

of AD. For decades, the “one drug for one target” strategy has

been undertaken in the development of treatment approachs,

but this was unable to conquer this multifactorial disease. It has

been suggested that pleiotropic agents which are capable of

simultaneously interacting with several pathologies, could be

acetylcholine metabolism, and prolong acetylcholine action at

the synapses. Butyrylcholinesterase (BuChE) also plays an

important role in the hydrolysis of acetylcholine in normal

Received: July 22, 2020

Accepted: September 21, 2020

eﬀective in AD treatment.

It has been reported that decreased choline acetyltransferase

activity, decreased cholinergic transmission, and cholinergic

neuron atrophy were correlated with dementia severity.

XXXX American Chemical Society

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Research Article

brains. In addition, BuChE was found to be associated with

The in vitro ChE inhibitory eﬀect of 9Q was evaluated using

Ellman’s method. Donepezil, the FDA approved cholinesterase

inhibitor for treating AD, was used as the positive control. The

IC₅₀values of 9Q on AChE and BuChE were determined as

1.68 μM and 16.34 μM, respectively (see Figure 2A).

Compound 9Q exhibited over 80% inhibition on both AChE

and BuChE at a concentration of 100 μM. It is noted that 9Q

outperformed donepezil in BuChE inhibition.

AD pathology, such as amyloid beta (Aβ) plaques, a major

hallmark of AD.

could be useful to enhance the symptomatic treatment of AD.

Among other hallmarks, Aβ is an important pathological

target due to its predominant role in AD pathology. In a

pathological condition, amyloid precursor protein (APP) is

catalyzed by β-secretase and γ-secretase to produce Aβ via the

amyloidogenic pathway. In physiological conditions, α-

secretase instead of β-secretase processes APP to form soluble

APPα fragment via the nonamyloidogenic pathway.

deposition of Aβ was thought to initiate a series of AD

pathologies, including senile plaques, neuroﬁbrillary tangles,

neuron death, and dementia. Accumulating evidence suggests

that amyloid oligomers are the major cause of neuro-

toxicity.

tion has been one of the desirable approaches to reduce Aβ

accumulation and toxicity in AD brains.

−11

Thus, inhibition of AChE and BuChE

In order to test the in vitro inhibitory eﬀect of 9Q on Aβ

aggregation, the MALDI-TOF MS instrument was used to

monitor residual monomeric Aβ42 in the aggregation

3,14

The

solutions. Tramiprosate, an Aβ aggregation inhibitor in

phase III clinical evaluation, was used as positive control. As

shown in Figure 2B, 9Q signiﬁcantly inhibited the aggregation

of Aβ42 compared with the vehicle control (F(2,15) = 15.25, p

= 0.0002). Compound 9Q (1 mM) showed 36.3% inhibition

of Aβ aggregation, outperforming tramiprosate at the same

concentration.

6,17

Designing inhibitors targeting amyloid aggrega-

18,19

Recently, the application of multitarget-directed ligands

MTDLs) has been reported as a very powerful and promising

To assess the toxicity of 9Q, MTT assay was used to

evaluate the cell viability of SH-SY5Y cells upon incubation

with the compound for 24 h. Compound 9Q showed minimal

toxicity even at 200 μM compared with the vehicle control

containing the same amount of DMSO (see Figure 2C).

2.2. Compound 9Q Penetrated the BBB and

Enhanced the Cerebral Acetylcholine Level in 3xTg-

AD Mice. To explore the ability of 9Q to cross the BBB, brain

deposition of 9Q was studied in 3xTg-AD mice. Following IP

dosing of 9Q (10 mg/kg), the plasma and brain samples were

harvested after 120 min. The concentrations of 9Q were found

to be 3.04 ± 1.54 ng/mL (mean ± SD, n = 10) in plasma and

(

0,21

approach to tackle AD.

AChEI-based MTDLs were

reported to modulate the disease by not only enhancing the

acetylcholine action but also inhibiting Aβ aggregation and

22−24

scavenging oxygen radicals.

In the present study, we

carried out extensive pharmacological evaluation of a

previously reported dual inhibitor of ChEs and Aβ aggregation,

Q (see Figure 1 ), using in vitro models and triple transgenic

4.65 ± 4.35 ng/g (mean ± SD, n = 10) in brain. To study the

in vivo ChE inhibitory eﬀect of the compound, the cerebral

acetylcholine level was determined after 120 min from IP

dosing (10 mg/kg 9Q). The cerebral acetylcholine levels in

PBS control and 9Q group were found to be 929.3 ± 70.55

ng/g (mean ± SEM, n = 10) and 1497 ± 110.2 ng/g (mean ±

SEM, n = 10), respectively (see Figure 3). This suggests that

Q treatment enhances the cerebral acetylcholine level in

3xTg-AD mice.

.3. Compound 9Q Ameliorated the Cognitive

Deﬁcits of 3xTg-AD Mice. Twenty-four female 3xTg-AD

mice were treated with designated dosing regimens for 4

weeks. We found that the body weight of the animals at week 4

changed slightly (with body weight changed by −8.4% for the

control group, −4.1% for the 9Q 3 mg/kg group, −4.3% for

the 9Q 10 mg/kg group, and −8.1% for the 9Q 30 mg/kg

group, compared with the initial weight), suggesting 9Q was

well tolerated. No signiﬁcant alterations of the appearance and

home-cage behavior were observed throughout the study.

MWM was used to evaluate the eﬀects of 9Q on the

cognitive deﬁcits of 3xTg-AD mice following 1 month

treatment. The MWM task can reﬂect the memory and spatial

learning of the mice. Compound 9Q treatment (30 (mg/kg)/

day, n = 8) signiﬁcantly reduced the latency time (time to

reach the platform) compared with the vehicle control on day

4 (F(3, 28) = 3.9, P = 0.019; see Figure 4A). Mice treated with

9Q (30 (mg/kg)/day) spent signiﬁcantly more time in the

target quadrant (F(3,28) = 3.263, P = 0.0429; see Figure 4B)

and performed signiﬁcant more platform crossings (F(3,28) =

Figure 1. (A) Chemical structure of 9Q. (B) Synthetic scheme of 9Q.

AD (3xTg-AD) mouse model. Cerebral accumulation of 9Q

and the acetylcholine content in mouse brains were

determined to evaluate, respectively, the blood−brain barrier

(

BBB) penetration ability of 9Q and the enhancement of

acetylcholine in vivo. The Morris water maze (MWM) was

used to assess the cognitive impairments of the 3xTg-AD mice

after 1 month treatment with 9Q. The expression levels of

diﬀerent AD pathological proteins in 9Q treated mouse brains

were studied using immunoassays, including enzyme-linked

immunosorbent assay (ELISA), Western blot assay, immuno-

histochemistry (IHC), and immunoﬂuorescence (IF). The

neuroprotective eﬀects of 9Q against tau hyperphosphoryla-

tion were demonstrated using the SH-SY5Y cell model.

.046, P = 0.0204; see Figure 4C) compared with the vehicle

. RESULTS

.1. Compound 9Q Suppressed in Vitro ChE Activity

and Aβ Aggregation and Exhibited Low Cytotoxicity.

treated mice during the probe trial. These results suggested

that 9Q treatment ameliorated the cognitive impairments in

the 3xTg-AD mice.

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Figure 2. Compound 9Q exhibited dual inhibition of ChE activity and Aβ42 aggregation and low cytotoxicity. (A) Compound 9Q suppressed

AChE and BuChE activities. The IC₅₀value of 9Q on AChE or BuChE was determined using the Ellman’s method. (B) Calculated inhibition

percent of the compounds (1 mM) on Aβ42 aggregation at 12 h from initiation. TP represents tramiprosate. (C) Cell viability of SH-SY5Y cells in

the presence of 9Q. All data are presented as the mean ± SEM (n = 2 for panel A, n = 6 for panels B and C). Signiﬁcance was analyzed by one-way

ANOVA (for panel B) followed by Dunnett ’s multiple comparisons test using GraphPad Prism. *p < 0.01 compared with vehicle control group.

is localized in presynaptic vesicles of nerve cells) proteins

compared with the vehicle group (F(3,12) = 22.86, P < 0.0001

for PSD-95; F(3,12) = 35.50, P < 0.0001 for SYP). SYP and

PSD-95 in subiculum regions of the hippocampus were studied

by IF staining (see Figure 6C,D). It is noted that the

ﬂuorescent signal of SYP or PSD-95 (red) in vicinity of the

ﬂuorescent signal of nuclei (blue) indicated SYP or PSD-95

positive. The images of SYP and PSD-95 were semiquantiﬁed

by ImageJ software. From the relative integrated density

(

Figure 6E), though not statistically signiﬁcant, enhancements

of SYP and PSD-95 in 9Q treated mice were observed. These

results suggested that 9Q exerted neuroprotective eﬀects

through enhancing the expression of synaptic proteins.

.6. Compound 9Q Decreased APP Level by

Figure 3. Compound 9Q enhanced the cerebral acetylcholine level in

xTg-AD mice. After 120 min treatment, the cerebral acetylcholine

level was detected. All data are presented as the mean ± SEM (n =

0). Signiﬁcance was analyzed by unpaired t test with Welch’s

correction using GraphPad Prism. *p < 0.01 compared with vehicle

control group.

Regulating APP Processing in 3xTg-AD Mouse Brains.

We then further explored the amyloid precursor protein (APP)

and the processing protease level in mouse brains after one-

month treatment with 9Q by Western blotting. As shown in

Figure 7, 9Q treatment for one-month down-regulated APP

level in 3xTg-AD mouse brains compared with vehicle control

group (F(3,12) = 145.4, P = 0.0002). Meanwhile, ADAM17

2.4. Compound 9Q Reduced the Cerebral Amyloid

(

the regulated α-secretase of APP) and BACE (β-site amyloid

Burden. The Aβ42 level in brain homogenate guanidine

hydrochloride fraction, which reﬂected the deposition of

amyloid in brain, was detected by ELISA. As shown in Figure

precursor protein cleaving enzyme) were found to be up-

regulated and down-regulated, respectively, in 9Q treated

mouse brains (F(3,12) = 45.71, P = 0.0015 for ADAM17;

F(3,12) = 13.89, P = 0.0003 for BACE). The PSEN1 and

PSEN2 protein levels were increased in 9Q treated mice

compared with the vehicle treated mice (F(3,12) = 275.3, P <

A, 1 month treatment with 9Q (30 (mg/kg)/day)

signiﬁcantly decreased Aβ42 deposition in the brains of the

xTg-AD mice compared with that of the vehicle control

F(3,20) = 5.953, P = 0.0045). Cerebral senile plaques were

(

.0001 for PSEN1; F(3,12) = 6.692, P = 0.0066 for BACE).

detected by IHC. Reduced cerebral plaques in hippocampus

were also observed after one-month treatment with 9Q via

IHC staining (see Figure 5B). These results indicated that 9Q

treatment reduced the cerebral amyloid deposition in the

These observations suggested that 9Q decreased APP level by

promoting the nonamyloidogenic pathway and inhibiting the

amyloidogenic pathway.

2.7. Compound 9Q Alleviated Tau Pathology and

xTg-AD mice.

.5. Compound 9Q Mitigated Synaptic Dysfunction

Cdk5 Activity in 3xTg-AD Mice. Tau hyperphosphorylation

is one of the important pathological hallmarks of AD. We

detected phosphorylated tau in 3xTg-AD mice by IHC

staining. Treatment with 9Q reduced the phospho-tau

(Ser202, Thr205) in mouse brains compared with the vehicle

control (see Figure 8A). We also detected the tauopathy

related protein levels in the brains of 3xTg-AD mice that were

treated with 9Q or PBS vehicle. As shown in Figure 8B,C, we

in the Brains of 3xTg-AD Mice. To assess the ability of 9Q

to reduce synapse loss in 3xTg-AD mice, the levels of

postsynaptic and presynaptic proteins were investigated by

using Western blotting and immunoﬂuorescence (IF) staining.

As shown in Figure 6A,B, 9Q treatment elevated the

expression of postsynaptic density 95 (PSD-95) and

synaptophysin (SYP, an integral membrane glycoprotein that

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Figure 4. Compound 9Q ameliorated the cognitive deﬁcits in the MWM studies after one month treatment. (A) Latency to reach the escape

platform. (B) Time spent in the platform quadrant within 1 min test on Day 5. (C) Number of platform crossings within 1 min test on Day 5. (D)

Representative trajectories of the mice in the second acquisition study on day 4. All data are presented as the mean ± SEM (n = 8). Signiﬁcance

was analyzed by repeated-measures one-way ANOVA (for panel A) and one-way ANOVA (for panels B and C) followed by Dunnett’s multiple

comparisons test using GraphPad Prism. *p < 0.05 compared with vehicle treated Tg mice.

observed that 9Q treatment signiﬁcantly reduced the tau

protein phosphorylation (F(3,12) = 78.23, P < 0.0001 for p-tau

viability was then evaluated by MTT assay. As shown in Figure

9B, 9Q treatment signiﬁcantly enhanced the cell viability of the

OA-treated SH-SY5Y cells in a concentration-dependent

manner (F(3,20) = 21.14, p < 0.0001).

(Ser202, Thr205); F(3,12) = 246.6, P < 0.0001 for p-tau

Ser422); F(3,12) = 32.56, P < 0.0001 for p-tau (Thr212);

(

F(3,12) = 78.41, P < 0.0001 for p-tau (Ser262)). Moreover,

Q was found to reduce the cyclin-dependent kinase 5 (Cdk5)

protein level (F(3, 12) = 19.38, P = 0.0005; see Figures 8B,C).

.8. Compound 9Q Attenuated OA-Induced Tau

Hyperphosphorylation and Reduced Cdk5 Action in

SH-SY5Y Cells. To corroborate the inhibitory eﬀects of 9Q

on tau hyperphosphorylation and Cdk5 protein expression

level in vitro, we performed our investigations using SH-SY5Y

cell line. SH-SY5Y cells were incubated with okadaic acid

3. DISCUSSION

The multifactorial etiology of AD has led to research into

MTDLs as an emerging strategy to tackle this disease.

inhibition is regarded as the most common approach that is

available for AD treatment for now. The amyloid hypothesis is

one of the most prevailing theories for AD etiology and has

attracted considerable attention in AD drug discovery.

Recently, we have reported some dual inhibitors of ChE

activity and Aβ aggregation. We found that 9Q outperformed

donepezil in the inhibition of BuChE. It has been reported that

BuChE was widely expressed in AD brains and contributed to

27,28

ChE

29,30

(

OA) to induce tau hyperphosphorylation. As shown in Figure

A, 40 nM OA treatment induced phosphorylated tau in SH-

SY5Y cell, while 9Q treatment reversed tau phosphorylation.

We also observed signiﬁcantly decreased in Cdk5 and p35/25

protein expression levels in the 9Q treatment group (see

Figure 9A). Collectively, our results suggested that 9Q

inhibited tau hyperphosphorylation through the down

regulation of Cdk5 activity in SH-SY5Y cells. To evaluate

the neuroprotective eﬀects of 9Q, the OA treated SH-SY5Y

cells were incubated with 9Q (1, 2, or 5 μM) for 24 h. The cell

,31

the hydrolysis of acetylcholine.

Moreover, 9Q out-

performed tramiprosate, an Aβ aggregation inhibitor that

has entered phase III clinical trials, in suppressing Aβ

aggregation. It should be pointed out that the in vitro assay

to evaluate amyloid aggregation inhibition served to rank the

potency of the compounds with respect to tramiprosate. Our

assay appears to be sensitive at 1 mM compound

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Figure 5. Compound 9Q reduced brain amyloid burden (Aβ42 levels) following one month of treatment. (A) Levels of Aβ42 in the guanidine

hydrochloride soluble fraction of brain tissue were detected by ELISA. All data are presented as the mean ± SEM (n = 6). Signiﬁcance was analyzed

by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism. **p < 0.01, compared with vehicle treated 3xTg-AD

mice. (B) Representative images of hippocampus amyloid plaques detected by IHC. Scale bar 200 μm (for panels a−d) and 100 μm (for panels e−

h).

concentration, taking into consideration of the inhibitory

potency of tramiprosate. It is noted that at a concentration of 1

mM, 9Q exerted obvious toxicity in the SH-SY5Y cell line,

with cell viability only 27.5% (data not shown). However, 9Q

did not appear to be toxic in the SH-SY5Y cell line up to 200

μM in our cytotoxicity evaluation. Here we directed our

attention to the in vivo pharmacological evaluation of 9Q.

The 3xTg-AD mouse model used in the present study was

generated by co-microinjection with APP and tau transgenes

into single-cell embryos from homozygous PS1 (M146V)

ELISA and IHC. As shown in Figure 5, 9Q reduced the

amyloid burden in 3xTg-AD mice. From the above in vitro and

in vivo studies, 9Q was found to be a promising dual inhibitor

of ChE activity and Aβ aggregation in 3xTg-AD mouse model.

As AD is a multifactorial disease, modiﬁcation of a speciﬁc

pathology may not necessarily lead to symptomatic improve-

ments. We observed that one-month treatment with 9Q

ameliorated the cognitive deﬁcits in 3xTg-AD mice which

might be due to multifaceted roles of 9Q in AD pathologies.

AD is characterized as cognitive impairment and memory loss.

Synapses are considered as the neurological basis of memory

knockin mice. The 3xTg-AD mouse develops progressively

plaques and tangles and age-related synaptic dysfunction and

cognitive deﬁcits, which is a valuable model for AD therapeutic

37,38

and learning.

Various synaptic marker proteins, such as

39,40

PSD-95

and SYP, were reported to be related to memory

3,34

evaluation.

Prior to extensive in vivo studies, 9Q was

loss and cognitive deﬁcits and were reduced in AD brains.

evaluated for BBB penetration. We found that 9Q exhibited

detectable brain exposure in 3xTg-AD mice. Following a single

dose of 9Q (10 mg/kg) to 3xTg-AD mice, concentrations of

Among the neuropathological features of the disease, synapse

dysfunction correlated best with dementia. As shown in

Figure 6, 9Q mitigated synapse loss in the brains of 3xTg-AD

mice. Emerging evidence suggested that Aβ and tau

accumulation contributed to synapse loss and the spread of

Q were found to be 10.2 nM in plasma and 15.6 nM in brain

at 2 h. Based on literature and our previous report, the Aβ

concentrations in cerebrospinal ﬂuid (CSF) of human and

plasma in mouse were no more than 2 ng/mL and 20 ng/mL,

which are equivalent to less than 0.4 nM and 4 nM,

respectively. As shown in Figure 3, single-dose of 9Q enhanced

the cerebral acetylcholine level at 2 h, suggesting that 9Q could

reach the brain to modulate the cerebral metabolism of

acetylcholine.

2,43

pathology.

The lack of APP was found to be beneﬁcial in

the formation of synapse. In the present study, the reduced

APP level (Figure 7) and relieved tau pathology (Figure 8)

were also observed in 9Q treated 3xTg-AD mice.

In AD brains, APP is processed predominately by β-

secretase and γ-secretase via the amyloidogenic pathway. As

shown in Figure 7, the upregulation of ADAM17 (the

regulated α-secretase) and down regulation of BACE indicated

that 9Q treatment regulated APP processing by promoting the

nonamyloidogenic pathway and suppressing the amyloidogenic

pathway. It is noted that, PSEN1 (presenilin 1) and PSEN2

(presenilin 2), the core proteins of γ-secretase, were

upregulated in 9Q treated 3xTg-AD mice. Point mutations

in the PSEN genes increase APP cleavage resulting in excessive

Next, we tested the pharmacodynamics eﬀects of 9Q in the

xTg-AD mouse model. The Morris water maze (MWM) was

designed to investigate the spatial learning and memory of

rodents and was widely used in the neurocognitive treatment

studies. Using the MWM, we found that 9Q treatment

ameliorated the cognitive deﬁcits in 3xTg-AD mice (see Figure

). We then examined the Aβ42 deposition in mouse brains by

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Figure 6. Compound 9Q mitigated synaptic dysfunction in 3xTg-AD mouse brains following one month of treatment. (A) Representative Western

blot of SYP, PSD-95, and GAPDH from brains of treated mice (PBS vehicle control and 9Q treated at dosages of 3, 10, and 30 (mg/kg)/day). (B)

Corresponding densitometry value ratios as mean ± SEM (n = 4). Signiﬁcance was analyzed by one-way ANOVA followed by Dunnett’s multiple

comparisons test using GraphPad Prism. *p < 0.05, **p < 0.01, compared with vehicle treated 3xTg-AD mice. (C) Representative images of SYP in

subiculum region of hippocampus studied by IF. (D) Representative images of PSD-95 in subiculum region of hippocampus studied by IF. Scale

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

di ﬀ erence (p ≥ 0.05) was observed compared with vehicle treated 3xTg-AD mice.

Research Article

Figure 6. continued

bar 50 μm (for panels C and D). (E) Relative integrated density of the immunoﬂuoroescent signals of panels C and D expressed as mean ± SEM (n

4). Signiﬁcance was analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism. No signiﬁcant

Figure 7. Compound 9Q decreased APP level by regulating APP processing in 3xTg-AD mouse brains following one month of treatment. (A)

Representative Western blot of APP, ADAM17, BACE, PSEN1, PSEN2, and GAPDH from brains of treated mice (PBS vehicle control and 9Q

treated at dosages of 3, 10, or 30 (mg/kg)/day). (B) The corresponding densitometry value ratios were the mean ± SEM (n = 4). Signiﬁcance was

analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism. **p < 0.01, compared with vehicle treated

xTg-AD mice.

Aβ, which would lead to familial AD cases. According to our

experimental data, though increasing γ-secretase was expressed

in the brain (Figure 7), the amyloid burden was reduced

instead (Figure 5).

MWM studies revealed that 9Q treatment for 1 month

ameliorated the cognitive deﬁcits in 3xTg-AD mice. It is noted

that several pathological hallmarks of AD, namely, synapse

dysfunction, amyloidogenic processing of APP and hyper-

phosphorylation of tau protein, were mitigated after 1 month

treatment with 9Q in 3xTg-AD mice. Taken together, our

results suggested that 9Q demonstrated eﬃcacy in 3xTg-AD

mouse model, which may provide new insights into the

development of multifunctional agent to treat Alzheimer’s

disease.

Cdk5 is one of the cyclin-dependent kinases (Cdks), which

has been reported to be closely related to the pathogenesis of

AD. Upon pathologic stimulus, Cdk5 became hyperactive,

which led to aberrant hyperphosphorylation of tau and

neuroﬁlaments, resulting in neurodegeneration. Cdk5, as a

tau kinase whose activity can be induced by Aβ peptides,

connects the amyloid toxicity to tau hyperphosphorylation.

Aβ toxicity elevates the intraneuronal calcium concentration,

4. EXPERIMENTAL PROCEDURES

which activates calpains to enhance the cleavage of p35 to

.1. Chemicals and Cell Culture. Compound 9Q (Figure 1) was

p25. The p25 binding to Cdk5 induces aberrant activity of

Cdk5, which plays an important role in tauopathy in human

synthesized as previously described. Donepezil and tramiprosate

were purchased from Aladdin Co. (Shanghai, China). The human

neuroblastoma SH-SY5Y cell line was a gift from Prof. Duncan Leung

(Institute of Chinese Medical Sciences, University of Macau). The

SH-SY5Y cells were cultured in humidiﬁed incubator at 37 °C with

and mouse models. It is believed that Cdk5 may contribute

to protein misfolding, toxicity, and undesirable interactions.

Targeting Cdk5/p25 might be a useful approach for treating

% CO . The cell culture medium was Dulbecco’s modiﬁed Eagle’s

AD. Our results suggested that 9Q reduced the Cdk5 activity

on tau phosphorylation in 3xTg-AD mice (Figure 8). The

eﬀects of 9Q on Cdk5 and tau phosphorylation was studied in

SH-SY5Y cell line. Western blotting has shown that 9Q

signiﬁcantly attenuated the tau hyperphosphorylation induced

by OA (Figure 9A). Furthermore, the induction of Cdk5, p25,

and p35 proteins conﬁrmed the inhibitory eﬀects of 9Q on

Cdk5 activity. It has been shown that 9Q could protect SH-

SY5Y cells against the toxicity induced by OA (Figure 9B).

Clearly our studies manifested that the dual inhibition of ChE

activity and Aβ aggregation could modulate the phenotypes of

AD via multifaceted processes.

medium (DMEM, Gibco) containing 12% fetal bovine serum (FBS,

Gibco) and 1% penicillin−streptomycin.

.2. In Vitro AChE and BuChE Inhibition Assays. The

inhibition of AChE and BuChE was measured by the Ellman method

with modiﬁcations. All reagents were prepared in a 0.1 M phosphate

buﬀer at pH 8.0 with 1.6 mM MgCl and 100 mM NaCl. In 200 μL

AChE catalytic reaction system, 0.1 U/mL AChE (Sigma-Aldrich), 1

mM acetylthiocholine (ATC, Sigma-Aldrich), and 1.2 mM 5,5′-

dithiobis(2-nitrobenzoic) acid (DTNB, Sigma-Aldrich) were incu-

bated with 9Q at diﬀerent concentrations. In 200 μL BuChE catalytic

reaction system, 0.05 U/mL BuChE (Sigma-Aldrich), 5 mM

butyrylthiocholine (BTC, Sigma-Aldrich), and 6 mM DTNB were

incubated with 9Q at diﬀerent concentrations. The reactions in 96-

well plates were started by spiking the prewarmed ATC and BTC.

Spectral data was recorded at 412 nm at 37 °C for 120 min by a plate

reader (SpectraMax M5, Molecular Devices). All the experiments

In conclusion, it has been shown that 9Q, a dual inhibitor of

ChEs and Aβ aggregation, enhanced the cerebral acetylcholine

level and reduced the amyloid burden in 3xTg-AD mice.

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Research Article

Figure 8. Compound 9Q attenuated tau pathology and Cdk5 activity in 3xTg-AD mouse brains following one month of treatment. (A)

Representative images of hippocampus phospho-tau detected by IHC. Scale bar 200 μm (for panels a−d) and 100 μm (for panels e−h). (B)

Representative Western blot of p-Tau(Ser202, Thr205), p-Tau(Ser422), p-Tau(Thr212), p-Tau(Ser262), HT7, p25, Cdk5, and GAPDH from

brains of treated mice (PBS vehicle control and 9Q treated at dosages of 3, 10, or 30 mg/kg). (C) Corresponding densitometry value ratios as

mean ± SEM (n = 4). Signiﬁcance was analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism. *p <

.05, **p < 0.01, compared with vehicle treated 3xTg-AD mice.

were carried out duplicate. Reactions without inhibitors were used to

aﬀord 100% AChE or BuChE activities. IC₅₀values were derived from

the concentration−inhibition curve of the inhibitors by the GraphPad

Prism program.

incubated with 9Q was evaluated by the MTT assay. A concentration

of 20 mM 9Q was solubilized in DMSO as the stock solution. The

SH-SY5Y cells were inoculated into 96-well plates at 40 000 cells per

well containing 100 μL of medium. After 24 h, the culture medium

was refreshed with addition of compound 9Q at concentrations

varying from 0 to 200 μM, and the cells were incubated for another 24

h. Then, each well was refreshed with medium containing 0.5 mg/mL

MTT, and the cells were further incubated for 4 h at 37 °C. MTT

solution was discarded, and 100 μL of DMSO was added into each

well to enable complete solubilization of the formazan. The

absorbance was measured at 590 nm with a plate reader (BioTek).

Wells without cells were used as blanks and deducted as background

from each sample. Inhibition was expressed as percentage of control.

IC₅₀values were then derived from the concentration−inhibition

curves by GraphPad Prism program.

.3. In Vitro Aβ Aggregation Inhibition Assay. The self-

induced Aβ42 aggregation samples were prepared as previously

described with modiﬁcations. In brief, artiﬁcial Aβ42 peptides were

solubilized in structure-breaking organic solvent 1,1,1,3,3,3-hexa-

ﬂuoro-2-propanol (HFIP, Sigma-Aldrich) at a concentration of 1 mg/

mL to form the uniform Aβ42 monomer solution and then

lyophilized. The lyophilized Aβ42 peptides were solubilized in

.05% ammonium hydroxide at 200 μM as the stock solution. The

aggregation process in 20 μM Aβ42 peptide solution (in 20 mM

sodium phosphate, pH 7.4) was initiated by adding solution

containing 1 μM Aβ42 ﬁbril seeds and 0.1 M NaCl. Compound

9Q was then added, and the resulting solution was incubated without

To test the neuroprotective eﬀects of 9Q against tau hyper-

shaking at 37 °C.

The preparation of Aβ42 ﬁbril seeding solution was accomplished

by incubating 50 μM Aβ42 in 20 mM sodium phosphate buﬀer, pH

phosphorylation, the OA-induced SH-SY5Y cell hyperphosphoryla-

tion model was used. After the SH-SY5Y cells were inoculated into

96-well plates and incubated for 24 h, the culture medium was

refreshed with addition of both OA (40 nM) and 9Q (0, 1, 2, 5 μM at

one concentration) or none. After incubation for another 24 h, the

medium was replaced with MTT solution. The experiment and the

analysis were then performed as described before.

.4, and 0.1 M NaCl in an incubator at 37 °C with continuous shaking

at 80 rpm for 2 days. Then this 50 μM Aβ42 solution was sonicated

for 30 min to generate the Aβ ﬁbril seeds and stored at −20 °C for

further usage.

The MALDI TOF MS (Microﬂex, Bruker) was used to determine

the residual monomeric Aβ content in aggregating solutions. The

detailed analytical method has been reported, and readers are directed

4.5. Cell Culture and Cell Protein Preparation for Western

Blot. The SH-SY5Y cells were cultured in 10 cm Petri dish at

4 000 000 cells per dish in 10 mL of medium. After 24 h, the culture

medium was refreshed with the addition of both OA (40 nM) and 9Q

(0, 1, 2, or 5 μM at one concentration) or none. After incubation for

another 18 h, cells were harvested into 0.5 mL of ice-cold RIPA buﬀer

to the literature for methodological details.

.4. Cell Viability Evaluation by MTT Assay. To test the

cytotoxicity of the compounds, the viability of SH-SY5Y cells

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

.7. Brain Exposure of Compound 9Q in 3xTg-AD Mice.

.7.1. Animal Dosing and Sample Collection. Twenty 10-month old

male and female mice were fasted but allowed free access to water for

4 h before dosing. Compound 9Q was solubilized in PBS at 1 mg/

mL and intraperitoneally (ip) injected to mice (n = 10) at a dose of

0 mg/kg. PBS as the vehicle control was injected to mice at a dose of

0 mL/kg. Blood (20 μL) was sampled via caudal vein to heparinized

microcentrifuge tubes at a time point 120 min from dosing. The

animals were then immediately terminated by CO asphyxiation, and

their brains were collected and stored at −80 °C for further analysis.

Plasma was separated by centrifuging the blood sample at 1500g, 4

C, for 20 min and stored in −80 °C for further analyses.

.7.2. Detection of 9Q in Plasma and Brain Samples by LC-MS/

MS. Plasma and brain samples were prepared using the protocols

previously described and analyzed by LC-MS/MS (Waters, TQD)

as previously described. Readers are directed to the relevant

literature for methodological details.

Compound 9Q (m/z 299 > 130 as quantiﬁer ion and m/z 299 >

42 as qualiﬁer ion) and berberine (as internal standard, m/z 336 >

20 as quantiﬁer ion and m/z 336 > 278 as qualiﬁer ion) were

simultaneously monitored via positive ionization mode.

.8. Detection of Cerebral Acetylcholine. The brain samples in

section 4.7.1 were used to evaluate the cerebral acetylcholine content.

All LC-MS/MS conditions were the same as that for 9Q detection,

except that acetylcholine was monitored using m/z 146 > 87 as

quantiﬁer ion and m/z 146 > 60 as qualiﬁer ion.

.9. Compound 9Q Treatment in 3xTg-AD Mice. Twenty-four

female 3xTg-AD mice (10 months old) were separated randomly into

groups. Group 1 was the PBS vehicle control group and groups 2−4

were, respectively, low (3 mg/kg), middle (10 mg/kg), and high (30

mg/kg) 9Q-dosing groups. Compound 9Q was freshly prepared in

PBS buﬀer every day for dosing. These mice were dosed

intraperitoneally once a day for 5 days per week (ﬁve consecutive

dosing days followed by two rest days) for one month. Food and

water consumption was monitored periodically. Body weights were

recorded weekly.

.10. Morris Water Maze (MWM). After the 3xTg-AD mice were

treated with 9Q for one month, MWM tests were performed with the

ZS Dichuang (Beijing, China) MWM equipment containing a Xeye

Aba video tracking system to assess memory and learning as described

previously. Brieﬂy, mice were trained to locate the invisible

submerged platform (8 cm in diameter) in the MWM circular pool

(

120 cm in diameter and 60 cm in height). The 2860 Bt7200 video

Figure 9. Compound 9Q attenuated OA-induced tau hyper-

phosphorylation and reduced Cdk5 activity in SH-SY5Y cells. (A)

Representative Western blot of p-Tau(Ser202, Thr205), p-Tau-

tracking camera was mounted directly above the center of the pool to

monitor activity within the pool. The temperature (22 °C) and

decorations of the pool as well as the location of the platform were

maintained constant throughout the mice training period.

(

Ser422), p-Tau(Ser262), HT7, p25, Cdk5, and GAPDH from SH-

SY5Y cells treated with OA and 9Q. (B) Viability of SH-SY5Y cells in

the presence of OA and 9Q. Signiﬁcance was analyzed by one-way

ANOVA followed by Dunnett’s multiple comparisons test using

GraphPad Prism. *p < 0.05, **p < 0.01, compared with OA alone

treatment group, ##p < 0.01, compared with no-treatment group.

During the acquisition phase, mice were trained for 4 consecutive

days with 4 trials per day at 4 starting points (E, SE, NW, and N) to

ﬁnd the submerged platform in opaque water. When a mouse located

the platform in 60 s, the mouse could stay on the platform for 5 s

before being returned to its home cage. Otherwise, the mouse would

be gently guided to the platform and allowed to stay on it for 15 s.

The time required to ﬁnd the platform (escape latency) was recorded.

On day 5, the probe trial without the submerged platform was

performed. In the 60 s probe trial, the mouse was freely allowed to

explore in the pool. Time spent in the target quadrant where the

submerged platform was located and the time that the mouse passed

across the platform (platform crossings) were recorded by the

tracking system.

(

50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1%

Triton X-100, 0.1% SDS, pH 7.5) containing protease inhibitor

cocktail (S8830, Sigma) and phosphatase inhibitor cocktail

(

04906845001, Roche) and mixed by gently rotation for 30 min at

°C. The supernatant was collected for Western blot assay by

centrifuging the sample at 12 000g for 20 min at 4 °C.

.6. Animals. The homozygous 3xTg-AD mice (B6; 129-

Psen1tm1MpmTg (APPSwe, tauP301L) 1Lfa/Mmjax) were obtained

from the Jackson Laboratory (Bar Harbor, Maine, USA) and

maintained by the Animal Facility in the Faculty of Health Sciences

4.11. Brain Sample Preparation. After the MWM studies, the

mice were terminated by CO asphyxiation, and immediately their

brains were collected. The left-brain hemispheres were ﬁxed in 4%

paraformaldehyde (PFA) for 48 h and dehydrated in 30% sucrose for

72 h for immunostaining. The right-brain hemispheres were frozen in

liquid nitrogen and stored at −80 °C for later protein extraction. Brain

proteins were extracted as previously described with some

modiﬁcation. Brieﬂy, the right-brain hemisphere was homogenized

and fractionated into three parts (1) TBS-soluble fraction, (2) RIPA

soluble fraction, and (3) guanidine hydrochloride soluble fraction.

(

University of Macau). Twenty 10-month old 3xTg-AD mice (10

male and 10 female) were used in the study of 9Q brain exposure and

in vivo ChE inhibitory eﬀect. Twenty-four 10-month old female 3xTg-

AD mice were used in the 9Q treatment study. The animal study

protocols (#UMARE-AMEND-086 and #UMARE-AMEND-087)

were approved by the Animal Research Ethics Committee of the

University of Macau.

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

Complete contact information is available at:

Research Article

The brain hemisphere was homogenized in 4 vol (w/v) of ice-cold

TBS containing protease inhibitor cocktail (S8830, Sigma) and

phosphatase inhibitor cocktail (04906845001, Roche) with a high-

throughput tissuelyser (Scientz-192) at 15 times/second for 1 min in

a 4 °C room. Samples were centrifuged (16 000g, 30 min at 4 °C),

and the TBS-soluble fraction was collected and stored at −80 °C. The

pellet was then rehomogenized in 4 vol of ice-cold RIPA buﬀer

containing protease inhibitor cocktail and phosphatase inhibitor

cocktail at 25 times/second for 2 min and mixed gently by rotation at

Sections were then blocked in PBS with 3% BSA at room temperature

for 1 h and incubated with the primary anti-Aβ42 antibody (805502,

Biolegend) or primary anti-phospho-tau (Ser202/Thr205) antibody

(MN1020, ThermoFisher) in PBS with 3% BSA at 4 °C overnight.

Sections were then incubated with 3% hydrogen peroxide followed by

biotinylated secondary antibody (31800, ThermoFisher) for 30 min at

room temperature. In the above operations, sections were washed 2

times in PBS with 0.2% Triton X-100 for 5 min before commencing

the next incubation procedure. ABC kit (32020, ThermoFisher) and

DAB quanto (TA-060-QHDX, ThermoFisher) were applied to

visualize brain sections. Hematoxylin was used to counterstain for

phospho-tau detection. Images of the IHC sections were acquired

using an Olympus BX53 system microscope equipped with Olympus

DP73 digital camera.

4.14.2. Immunoﬂuorescence (IF). The IF was performed for SYP

and PSD-95 detection. After antigen retrieval, permeabilization,

blocking, and incubation with primary anti-SYP antibody (AB9272,

Millipore) or primary anti-PSD-95 antibody (3450s, Cell Signaling

Technology) in the same procedure as for phospho-tau detection

introduced in section 4.14.1, sections were incubated with the Alexa

Fluor 594 cross-adsorbed secondary antibody (A11012, Thermo-

Fisher) for 30 min at room temperature. In the above operations,

sections were washed 2 times in PBS with 0.2% Triton X-100 for 5

min before commencing the next incubation procedure. DAPI was

used to counterstain. Images of the sections were acquired using a

Nikon Ti-E ﬂuorescent microscope equipped with Nikon DS-Qi2

digital camera. Signals of SYP and PSD-95 positive in IF images were

quantiﬁed by ImageJ software.

°C for 30 min. Samples were centrifuged (16 000g, 30 min at 4 °C),

and the RIPA soluble fraction was collected for Western blot assay.

The pellet was then resuspended in 5 M guanidine hydrochloride/50

mM Tris-HCl, pH 7.5, to 150 mg/mL (based on pellet weight) and

mixed by rotation for 2 h at room temperature. Samples were

centrifuged (16 000g, 30 min at 4 °C), and the guanidine

hydrochloride fraction was collected and frozen.

.12. Enzyme-Linked Immunosorbent Assay (ELISA). The

guanidine hydrochloride soluble fraction of the brain tissue was used

for detection of Aβ42 deposition in brain. The mouse speciﬁc Aβ42

ELISA kit was obtained from Invitrogen (KMB3441). The experi-

ment was operated as the product manual described. Plates were

analyzed using a plate reader (SpectraMax M5, Molecular Devices).

.13. Western Blot Assay. The brain homogenate RIPA soluble

fraction and cell lysates were separately used to study the expression

levels of various proteins. Protein concentrations were assessed by

Pierces BCA Protein Assay Kit (Thermo Fisher Scientiﬁc). All

samples were mixed with 5× sample buﬀer (250 mM Tris-HCl, pH

.8, 8% SDS, 50% glycerol, 500 mM dithiothreitol (DTT), and 0.02%

bromophenol blue) and boiled for 5 min at 100 °C. Samples

containing an equal amount of protein were separated by SDS-PAGE

and transferred onto poly(vinylidene diﬂuoride) (PVDF) membrane.

The membrane was blocked in 5% bovine serum albumin (BSA) for 2

h and incubated with desired primary antibodies overnight, followed

by anti-rabbit or anti-mouse HRP-conjugated secondary antibody

4.15. Statistical Analysis. Statistical analyses were carried out

using GraphPad Prism (version 6.02, San Diego, CA, USA). The 9Q

content data was expressed as mean ± standard deviation (SD), and

other data was expressed as mean ± standard error of the mean

(SEM). Unpaired t test with Welch’s correction was used for cerebral

acetylcholine level data analysis, repeated-measures one-way variance

(ANOVA) followed by Dunnett’s multiple comparisons test was used

to analyze the data obtained from MWM study, and one-way ANOVA

followed by Dunnett’s multiple comparisons test was used for other

data analysis. Data with p ≤ 0.05 was considered signiﬁcant.

(

Cell Signaling Technology) for 2 h. Membranes were ﬁnally

visualized in a ChemiDoc MP Imaging System (Bio-Rad) after 2

min incubation in ECL Substrate (6883S, Cell Signaling Technology).

The primary antibodies used for detection were as follows: anti-APP

antibody (2452s, Cell Signaling Technology), anti-ADAM antibody

(

ab39162, Abcam), anti-BACE antibody (5606p, Cell Signaling

AUTHOR INFORMATION

Corresponding Author

■

Technology), anti-presenilin 1 antibody (5643P, Cell Signaling

Technology), anti-presenilin 2 antibody (9979p, Cell Signaling

Technology), anti-PSD 95 antibody (3450s, Cell Signaling Technol-

ogy), anti-synaptophysin (AB9272, Millipore), anti-phospho-tau

Kin Yip Tam − Faculty of Health Sciences, University of Macau,

Taipa, Macau, China; orcid.org/0000-0001-5507-8524;

Email: kintam@um.edu.mo

(

Ser202/Thr205) antibody (MN1020, ThermoFisher), anti-phos-

Authors

Yaojun Ju − Faculty of Health Sciences, University of Macau,

Taipa, Macau, China

Signaling Technology), anti-Cdk5 antibody (2506s, Cell Signaling

Technology), anti-GAPDH antibody (5174s, Cell Signaling Technol-

ogy). Western blot images were analyzed by ImageJ software

Harapriya Chakravarty − Faculty of Health Sciences, University

of Macau, Taipa, Macau, China

https://pubs.acs.org/10.1021/acschemneuro.0c00464

(

National Institutes of Health, USA) to quantify the band signal

intensity.

.14. Immunostaining. Coronal sections of the left hemisphere

of mouse brains were collected using a cryotome cryostat (CM5030,

Leica). The 20 μM thick coronal sections were inserted onto slides for

Aβ42 and phosphorylated tau detection by IHC, while SYP and PSD-

Author Contributions

Y.J. performed experiments, including animal work, biochem-

istry, IHC, and IF, and analyzed the data. Y.J. and K.Y.T.

discussed the results and composed and edited the manuscript.

H.C. performed chemical synthesis. K.Y.T. supervised all

phases of the study.

5 detections were accomplished by IF. The slides were air-dried at

room temperature overnight and then stored at −80 °C until further

usage. At the beginning of the immunostaining, slides were all ﬁxed in

cold acetone at −20 °C for 15 min.

.14.1. Immunohistochemistry (IHC). The IHC study was

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Dr. Xiaohui Hu for helpful suggestions and

discussion throughout this project. We thank Prof. Duncan

Leung (ICMS, University of Macau) for a sample of SH-SY5Y

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

Baldeiras, I. (2017) Association between butyrylcholinesterase and

Research Article

cells. We thank the Animal Research Core and Biological

Imaging & Stem Cell Core at Faculty of Heath Sciences,

University of Macau, for technical support. This work was

funded by University of Macau (File no. MYRG2016-00102-

FHS).

cerebrospinal fluid biomarkers in Alzheimer ’s disease patients.

Neurosci. Lett. 641, 101−106.

(12) Selkoe, D. J., and Hardy, J. (2016) The amyloid hypothesis of

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ABBREVIATIONS

(

■

AD, Alzheimer’s disease; Aβ, amyloid beta; AChE, acetylcho-

linesterase; BuChE, butyrylcholinesterase; APP, amyloid

precursor protein; BBB, blood−brain barrier; NMDAR, N-

methyl-D-aspartate receptor; MTDL, multitarget-directed

ligand; ATC, acetylthiocholine; DTNB, 5,5′-dithiobis(2-nitro-

benzoic) acid; BTC, butyrylthiocholine; HFIP, 1,1,1,3,3,3-

hexaﬂuoro-2-propanol; MALDI, matrix-assisted laser desorp-

tion ionization; CHCA, α-cyano-4-hydroxycinnamic acid;

TFA, triﬂuoroacetic acid; OA, okadaic acid; MWM, Morris

water maze; PFA, paraformaldehyde; DTT, dithiothreitol;

PVDF, poly(vinylidene diﬂuoride); BSA, bovine serum

albumin; IHC, immunohistochemistry; SYP, synaptophysin;

PSD-95, postsynaptic density 95; IF, immunoﬂuorescence;

APP, amyloid precursor protein; BACE, β-site amyloid

precursor protein cleaving enzyme; Cdk5, cyclin-dependent

kinase 5

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