The phosphodiesterase 5 inhibitor, KJH-1002, reverses a mouse model of amnesia by activating a cGMP/cAMP response element binding protein pathway and decreasing oxidative damage

Article abstract of DOI:10.1111/bph.14377

Background and Purpose: Inhibition of PDE5 improves synaptic plasticity and memory via enhancing cGMP expression, thus activating the cGMP/cAMP response element binding protein (CREB) signalling pathway. This study investigated the effects of a PDE5 inhibitor on scopolamine-induced cognitive dysfunction, using memory-related behavioural tests and biochemical assays. Experimental Approach: In mice were pretreated with PDE5 inhibitor, amnesia was induced by scopolamine. The learning and memory abilities of mice were tested using the Morris water maze test, the Y-maze test, the passive avoidance test and the novel object recognition test in sequence. Expression of memory-related bio-molecules and oxidative stress parameters in brain tissue were measured using Western blot and spectrophotometry respectively. Key Results: KJH-1002, a novel and potent inhibitor of PDE5 (IC₅₀ 0.059?±?0.04?nmol·L^?1), was synthesized. In the behavioural tests, it markedly improved the memory performance impaired by scopolamine, indicating a restoration of cognitive function in the mice. Moreover, KJH-1002 increased cGMP levels in the cortex and the scopolamine-reduced expression of phosphorylated CREB, Levels of ERK 1/2, Akt and brain-derived neurotrophic factor in the cortex and hippocampus were restored by KJH-1002 treatment. In addition, KJH-1002 administration increased the activities of SOD, glutathione peroxidase and glutathione reductase, and decreased the level of malondialdehyde. Conclusion and Implications: KJH-1002 restored cognitive function in scopolamine-induced amnesia mice by activating the cGMP/CREB signalling pathway and attenuating oxidative stress. The beneficial effects of KJH-1002 on cognition indicate its potential as a therapeutic candidate for Alzheimer's disease.

Full text of DOI:10.1111/bph.14377

A novel synthetic phosphodiesterase 5 inhibitor, KJH-1002, ameliorates
scopolamine-induced cognitive impairments in mice by activating the
cGMP/CREB signaling pathway and attenuating oxidative damage
Lijun Zhang1,2,#, Jae Hong Seo3,#, Huan Li1,2, Ghilsoo Nam2,4,*, Hyun Ok Yang1,2,*
1Natural Products Research Center, Korea Institute of Science and Technology, 679
Saimdang-ro, Gangneung, Gangwon-do, 25451, Korea
2Division of Bio-medical Science and Technology, KIST School, Korea University of Science
and Technology, Seoul 02792, Republic of Korea
3Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The
Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon, Gyeonggi-do 14662,
Republic of Korea
4Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and
Technology, Seoul, 02792, Korea
#These authors contributed equally to this paper and they are the co-first authors
*Corresponding Author:
Ghilsoo Nam. E-mail: gsnam@kist.re.kr. Tel: +82- 02-958-5165
Hyun Ok Yang. E-mail: hoyang@kist.re.kr. Tel: +82-33-650-7101. Fax: +82-33-650-7199.
This article has been accepted for publication and undergone full peer review but has not
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.14377
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BACKGROUND AND PURPOSE
Inhibition of PDE5 has been demonstrated to improve synaptic plasticity and memory via
enhancing of cGMP expression, thus activating the cGMP/CREB signaling pathway. This
study aimed to investigate the ameliorating effect of PDE5 inhibitor on scopolamine-induced
cognitive dysfunction using memory-related behavioral tests and biochemical assays.
EXPERIMENTALAPPROACH
After the mice were pretreated with PDE5 inhibitor, amnesia was induced by scopolamine
administration. The learning and memory abilities of mice were tested using the Morris water
maze test, the Y-maze test, the passive avoidance test and the novel object recognition test in
sequence. Expression of memory-related bio-molecules and oxidative stress parameters in
brain tissue were measured using western blot and spectrophotometry, respectively.
KEY RESULTS
KJH-1002, a novel inhibitor of phosphodiesterase 5 (PDE5), was synthesized (IC50 of 0.059 
0.04 nmol·L-1), and it markedly improved the memory performance impaired by scopolamine
in the behavioral tests, indicating a restoration of cognitive function in the mice. Moreover,
KJH-1002 increased the cGMP level in the cortex, the scopolamine-reduced expression of
phosphorylated cAMP response element binding protein (CREB), extracellular-regulated
kinase 1/2 (ERK 1/2), protein kinase B (Akt) and brain-derived neurotrophic factor (BDNF)
in the cortex and hippocampus were reversed by KJH-1002 treatment. In addition, KJH-1002
administration increased the activities of superoxide dismutase (SOD), glutathione peroxidase
(GPx) and glutathione reductase (GR), and decreased the level of malondialdehyde (MDA).
CONCLUSION AND IMPLICATIONS
KJH-1002 restored cognitive function in scopolamine-induced amnesia mice by activating
the cGMP/CREB signaling pathway and attenuating oxidative stress. The beneficial effect of
KJH-1002 on cognition suggests its potential as a therapeutic candidate for Alzheimer’s
disease.
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Abbreviations
BDNF, brain-derived neurotrophic factor; CREB, cAMP response element binding protein;
SOD, superoxide dismutase; GPx, glutathione peroxidase; GR, glutathione reductase; MDA,
malondialdehyde; MWM, Morris water maze; NOR, novel object recognition
Table of Links
TARGETS
Kinasesa
LIGANDS
Donepezil
Akt (Protein kinase B)
Mitogen-activated protein kinase 1
Mitogen-activated protein kinase 3
Scopolamine
Sildenafil
Enzymesb
Acetylcholinesterase
Phosphodiesterase 5A
Introduction
The cyclic nucleotides, cAMP and cGMP are second messengers that can modulate signal
transduction by activating the phosphorylation of other enzymes or transcription factors in a
variety of biological systems. Phosphodiesterases (PDEs) which comprise 11 subtypes,
hydrolyze cAMP and/or cGMP and thereby disturb the cAMP/cGMP signaling pathway
(Hutson et al., 2011; Bollen et al., 2014). Phosphodiesterase 5 (PDE5), the selective enzyme
that breaks down cGMP, is expressed in a variety of cognitive function-related brain regions
(Van Staveren et al., 2003). Numerous studies have demonstrated that cyclic nucleotides are
involved in learning and memory and cGMP plays a key role in early memory consolidation,
while the late phase memory consolidation process is mainly attributed to cAMP (Bollen et
al., 2014). Inhibition of PDE5 elevates cGMP which activates the phosphorylation of CREB,
thereby enhances synaptic plasticity and cognition (Puzzo et al., 2009; Cuadrado-Tejedor et
al., 2011; Garcia-Osta et al., 2012).
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Alzheimer’s disease (AD), defined as the progressive and irreversible decline of
cognitive function, is the most prevalent form of dementia. Deficits in memory and learning
are typical features of AD patients, and cognitive impairment is attributed to remarkable
cholinergic neuron loss and decreased acetylcholine level via acetylcholinesterase (AChE)
(Nedaei et al., 2016). Oxidative stress is another crucial neurodegenerative disorder factor in
AD patients (Gella, 2009; Bonda et al., 2010). Scopolamine, a non-selective muscarinic
antagonist, disturbs learning and memory via blocking the effects of neurotransmitter
acetylcholine (Gautam et al., 2016; Kulshreshtha et al., 2016). It also induces neurotoxicity
by altering the levels or activities of antioxidant enzymes and abnormal expression of
proteins (Li et al., 2016; Venkatesan et al., 2016).
Accumulating evidence has demonstrated administration of phosphodiesterase 5
inhibitors, such as sildenafil and vardenafil, enhance cognitive function by increasing the
cGMP levels in the brains of various animal models (Prickaerts et al., 2002; Rutten et al.,
2005; Puzzo et al., 2009). Sildenafil improves memory consolidation in object recognition
tasks in unimpaired rats (Prickaerts et al., 2005) and mice (Rutten et al., 2005). Another study
(Cuadrado-Tejedor et al., 2011) reported that sildenafil ameliorates memory in the Morris
water maze (MWM) task in an AD mouse model of Tg2576. Moreover, scopolamine
impaired learning and memory in 14-unit T-maze task is attenuated by sildenafil
administration (Devan et al., 2004). Cognitive function is regulated by complicated signaling
pathways, and BDNF, a downstream target protein of CREB, plays a key role in memory
plasticity and formation and facilitates hippocampal cell proliferation (Erickson et al., 2010).
Moosavi et al. (2012) suggested that hippocampal extracellular-regulated kinases 1/2 (ERK
1/2) and activated protein kinase B (Akt) play important roles in regulating proteins in
synaptic plasticity and different kinds of learning and memory. Furthermore, Akt and CREB
are described as enhancers of neuronal survival, as they exert anti-apoptotic effects on
neuronal cells, and they can be regulated by cGMP (Takada-Takatori et al., 2006; Calabrese
et al., 2007).
Novel compound can be synthesized by the modification of heterocyclic nucleus, rigid
co-planarity or variation of bioisostere. To develop potent and selective PDE5 inhibitor, we
tried to synthesize a novel compound by bioisosteric transformation based on the structure of
sildenafil and test its inhibiting effects on PDE5. We then utilized a scopolamine-induced
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amnesia mouse model to mimic the cognitive dysfunction symptoms in AD patients. To
evaluate the enhancing effects of this synthesized compound on memory and learning, the
mice were subjected to a series of behavioral tests. Furthermore, to elucidate the underlying
mechanisms of this compound improving cognitive function, biochemical analysis were
carried out using the brain tissues.
Methods
Animals
Imprinting control region (ICR) mice (8 weeks-old males) were purchased from Orient Bio
Inc. (Seongnam, Korea). Four mice were housed in each cage (30 x 18.5 x 13 cm), with wood
shaving bedding and nesting material, and they were allowed unlimited access to food and
water. The animals were housed under constant conditions (12 h light/dark cycle, lights on
from 6:00 to 18:00; temperature 23 ± 1°C: humidity 50 ± 10%). To habituate the mice to the
ambient environment prior to the experiments, they remained under the above mentioned
conditions for one week after arrival. All the experimental protocols executed in this study
were in compliance with the Korea Institute of Science and Technology Animal Care
Committee guidelines, and the animal studies are reported in compliance with the animal
research: reporting of in vivo experiments (ARRIVE) guidelines (Kilkenny et al., 2010;
McGrath and Lilley, 2015). We took all effort to minimize the number of mice used and
relieve their suffering.
Grouping and pretreatment
Forty mice were randomly divided into five groups (8 mice per group), and all the mice in
each group were subjected to a 7-day pretreatment period. Mice in the normal control group
(Control), and in the scopolamine control group (Vehicle) were administered saline (p.o.).
Mice in the positive control group (DON) were administered donepezil (4 mg·kg-1, p.o.), and
mice in sample-treated groups (KJH-1002-10 and KJH-1002-20) were administered 10 or 20
mg·kg-1 KJH-1002 (p.o.), respectively. All mice received the gavage with saline, donepezil or
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KJH-1002 once daily in a volume of 10 mL·kg-1. The group size for each experiment was
determined by referring to previous studies (Moosavi et al., 2012; Li et al., 2016; Nedaei et
al., 2016) which reported similar protocols to those of the present study, at least five samples
is needed to detect the significant difference between groups. Therefore, we decided to use 6
or 8 samples for each experimental group (in vitro or in vivo).
Preparation of KJH-1002
Synthesis of 4-(2-Ethoxyphenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidine-7-thione (2)
5-(2-Ethoxy-phenyl)-1-methyl-3-N-propyl-1,6-dihydro-pyrazolo[4,3-d]pyrimidine-7-one (1)
593 mg (1.186mmol) and Lawesson's reagent (P4S10) 400 mg (0.953mmol) in toluene (7 mL)
were refluxed for 2h. The solvent was concentrated by a rotary evaporator and the residual
solution was purified using column chromatography (eluent-chloroform:EA = 10:1) to yield
the title compound as a yellow solid (547 mg, 89%).
1H-NMR (300 MHz, CDCl3): δ12.64 (bs, 1H), 8.46 (d, J = 7.9 Hz, 1H), 7.45 (t, J = 8.4 Hz,
1H), 7.13 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 4.52 (s, 3H), 4.18 (t, J = 6.5 Hz, 2H),
2.94 (t, J = 7.4 Hz, 2H), 2.6 (m, 2H), 1.85 (m, 2H), 1.18 (t, J = 7.4 Hz, 3H), 1.02 (t, J = 7.4
Hz, 3H); 13C-NMR (75 MHz, CDCl3): δ 171.90, 157.17, 148.34, 146.52, 134.74, 133.14,
132.63, 131.14, 122.18, 119.33, 113.06, 71.80, 39.71, 28.05, 22.90, 22.70, 14.45, 11.27; mp:
110-111 °C.
Synthesis of 5-[2-Ethoxy-5-(4-methyl-piperazine-1-sulfonyl)-phenyl]-1-methyl-3-propyl-
1,6-dihydro-pyrazolo[4,3-d]pyrimidine-7-thione (3)
4-(2-Ethoxyphenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-7
thione (4.26 g, 0.0130 mol) was added to chlorosulfonic acid (8.63 mL, 0.123 mol) in a
portionwise manner in an ice bath, followed by the addition of thionyl chloride (1.42 mL,
0.0195 mol). The mixture was stirred at room temperature overnight and then poured on ice
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(40 g) dropwise. The yellow solid was filtered and washed with water, and the wet solid was
suspended in EtOH (55 mL), prior to 1-methylpiperazine (5.09 mL, 0.0454 mol) being added
in an ice bath. The mixture was stirred at room temperature overnight, and the solvent was
removed under reduced pressure. The residue was added to a 6N aqueous solution of NaOH
(15 mL) and extracted with CH2Cl2 (40 mL). The organic layer was washed with brine (10
mL), dried with anhydrous MgSO4, filtered and evaporated to dryness. The crude product
was purified with crystalize in EhOH (40 mL) to yield the title compound as a yellow solid
(5.75 g, 90. 3%).
1H-NMR (300 MHz, CDCl3): δ 12.41 (bs, 1H), 8.82 (d, J = 2.3 Hz, 1H), 7.82 (dd, J = 8.7 Hz,
J' = 2.3 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 4.51 (s, 3H), 4.41 (q, J = 6.9 Hz, 2H), 3.10 (bs, 4H)
2.95 (t, J = 7.5 Hz, 2H), 2.50 (bs, 4H), 2.27 (s, 3H), 1.86 (m, 2H), 1.72 (t, J = 6.9 Hz, 3H),
1.02 (t, J = 7.4 Hz, 3H). 13C-NMR (75 MHz, CDCl3): δ 172.09, 159.83, 146.81, 146.43,
134.23, 132.64, 132.35, 131.03, 129.29, 120.09, 113.56, 66.81, 54.38, 46.31, 46.09, 39.74,
27.97, 22.53, 15.05, 14.41. IR (KBr pellet, cm-1) 3278, 2940, 2790, 1538; mp: 181-181.5 °C
Synthesis of 5-[2-Ethoxy-5-(4-methyl-piperazine-1-sulfonyl)-phenyl]-1-methyl-3-propyl-
1,6-dihydro-pyrazolo[4,3-d]pyrimidine-7-thione salt with 3-carboxy-3-hydroxy-
pentanedioic acid (KJH-1002)
Citric acid was dissolved in 26.4 mL of an acetone/water solution (10:1), and the
undissolved material was filtered. Compound 5-[2-Ethoxy-5-(4-methyl-piperazine-1-
sulfonyl)-phenyl]-1-methyl-3-propyl-1,6-dihydro-pyrazolo[4,3-d]pyrimidine-7-thione
(5.74 g, 0.0117 mol) was dissolved in refluxing acetone (72.5 mL) and thionyl chloride
(1.42 mL, 0.0195 mol), and undissolved material was filtered. The two obtained solutions
were mixed and refluxed for 1.5 h and then gradually cooled to room temperature. The
resulting solid was filtered and vacuum-dried to yield title compound as a yellow-white
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solid (7.75 g, 97.0%, KJH-1002(C22H36N6O3S2)). The brief synthetic process is shown in
Figure 1.
1H-NMR (300 MHz, DMSO-d6): δ 13.55 (bs, 1H), 7.94 (d, J = 2.4 Hz, 1H), 7.87 (dd, J =
8.8 Hz, J' = 2.4 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 4.45 (s, 3H), 4.24 (q, J = 6.9 Hz, 2H), 2.97
(bs, 4H) 2.82 (t, J = 7.4 Hz, 2H), 2.67 (ABq, J = 15.5 Hz, 4H), 2.55 (bs, 4H), 2.27 (s, 3H),
1.76 (m, 2H), 1.35 (t, J = 6.9 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H). 13C-NMR(75 MHz, DMSO-
d6): δ 175.87, 172.86, 172.13, 161.11, 148.53, 145.63, 134.28, 132.84, 132.68, 130.82,
127.13, 123.56, 114.28, 73.04, 65.97, 54.02, 46.11, 45.55, 43.86, 41.21, 40.02, 27.81, 22.42,
15.16, 14.65, IR (KBr pellet, cm-1) 3282, 2958, 1722, 1572; mp: 179-180 °C.
Assay to measure PDE5 activity inhibition by KJH-1002
Inhibition of the activity of PDEs 1, 3, 4, 5, 6 was determined using a modified version of the
method provided by Thompson et al. (1971). Briefly, the enzyme reaction mixtures (total
volume of 100 L: PDE enzyme 3~10 L, 10 nM CaCl2, 20 M calmodulin, 1  Ci/L
[3H]cGMP, 1  Ci/L [3H]cAMP, 0.01 nM~1M KJH-1002, 50 mM Tris-HCl buffer (pH 7.4),
15 mM MgCl2, and distilled water) were incubated in a water bath at 30 °C for 30 min. After
boiling for 2 min, ice-cold water (0.5 mL) was added, and the resulting solution was applied
to a DEAE-Sephacel A-25 anion exchange column. Aqueous scintillation fluid (20 mL) was
added to each solution, and the radio-activity was measured by a  counter. The KJH-1002
compound was used by diluting a solution of dimethyl sulfoxide (final volume 2%, v/v). The
IC50 values were calculated by a sigma plot; sigmodal curves of the remaining enzyme
activity (% of control) versus the log compound concentration graph were obtained, and the
point at which 50% activity remained was determined by linear regression.
Morris water maze test
After pretreatment for 7 days, the MWM test was employed to evaluate spatial memory and
learning in the mice. The test was performed as described by Morris et al. (1984). In brief, the
apparatus consisted of a circular pool (120 cm in diameter, 60 cm in height) with a featureless
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inner surface and an automatic tracking system (Ethovision System, Noldus, Wageningen,
The Netherlands). The circular pool was equally divided into four quadrants and filled with
water (45 cm deep, 23 ± 1°C), and a white, non-toxic dye (Blick Premium Grade Tempera
White Gallon, 00011-1009, BLICK, USA) was added to the pool to make the water opaque.
A circular platform (8 cm in diameter) was placed in the center of the target quadrant, and the
platform was submerged 1 cm below the water surface to be invisible to the mice. A black
curtain was arranged around the pool to isolate interference from irrelevant visual cues, and
four cues of assorted colors and shapes were fixed on the curtain opposite of each quadrant as
the only way for the mice to discriminate their position.
The MWM test consisted of an acquisition section and a retention section, and the test
was carried out for 6 consecutive days; the acquisition section was performed for the first 5
days. The mice were allowed to swim in the water while searching for the hidden platform for
90 s. If the mice found the hidden platform successfully, they were allowed to remain on the
platform for at least 5 s. Mice that failed to locate the platform in 90 s, were placed on the
platform for at least 5 s and then returned to their cage by the operator. Each mouse was
subjected to 3 trials per day at intervals of 30 min. For each trial, the mice began swimming
from different position and faced the inner wall of the pool. The time the mice spent
searching for the platform was recorded as the escape latency. For the mice unable to find the
platform, a latency of 90 s was recorded. On the day after the acquisition section, all mice
were subjected to a retention section in which the hidden platform was removed. The mice
were allowed to swim in the pool for 120 s, and the time spent in the target quadrant where
the platform had been fixed during the acquisition test was recorded. The mice were treated
with saline (Control and Vehicle), 4 mg·kg-1 donepezil (DON) 1 h before the trial (p.o.), and
scopolamine was injected 30 min prior to the trial (i.p.); for the KJH-1002-10 and KJH-1002-
20 groups, 10 or 20 mg·kg-1 KJH-1002 was administered 5 min prior to the trial, respectively
(p.o.).
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Y-maze test
The Y-maze was conducted to test the spontaneous alternation behavior as an estimation of
short-term memory. Briefly, the apparatus consisted of a three-arm maze with equal angles.
The identical arms were made of polyvinyl plastic, and the floors and walls were 30 cm in
length, 5 cm in width and 12 cm in height. The mice were allowed to begin the test facing the
terminal wall of one arm, and the mouse movements in the maze were recorded by a camera
fixed 1.5 m above the apparatus for duration of 5 min. Arm entry was defined as a mouse
placing all four paws in any arm completely, and the total number of arm entries and
sequence of arm entry (e.g., ABC, BCA or CAB) were counted manually. The actual
alternation behaviors were defined as entries in which the mice went into three consecutive
arms in sequence, such as ABC, BCA or CAB, but not ABA, BCB or CAC. An ethanol
solution (10%, v/v) was used to remove the residual odors between mice. One hour prior to
the Y-maze test, mice in Control, Vehicle and DON groups were administered saline, saline or
donepezil 4mg·kg-1, respectively (p.o.). After 30 min, all mice except for those in Control
group were administered 1 mg·kg-1 scopolamine (i.p.). Mice in the KJH-1002-10 and KJH-
1002-20 groups were administered 10 or 20 mg·kg-1 KJH-1002 (p.o.), respectively, 25 min
after the scopolamine injection. Thirty minutes after the scopolamine injection, the Y-maze
test was administered. The ratio of actual alternation behavior, calculated as the % alternation
= [(number of actual alternations) / (number of total arm entries-2)] x 100, was used as an
index to estimate short-term memory. The total number of mouse arm entries was analyzed to
assess the locomotor activity.
Passive avoidance test
The passive avoidance test was conducted in an apparatus with two identical compartments
(20 x 24 x 30 cm). One compartment was bright (illuminated by an 8 W lamb), and the other
compartment was dark, and equipped with 2-mm (diameter) stainless steel rods delivered
electrical shock to the mice. The two compartments were separated by a wall with a guillotine
door (5 x 5 cm). The passive avoidance test was performed during two consecutive days. And
the acquisition trial took place on day one. The mice were placed in illuminated compartment,
and after exploring for 30 s, the guillotine was opened and the mice were allowed to freely
enter the dark compartment. The door was closed once the mice entered the dark
compartment with no hesitation, and an electrical shock (0.3 mA, 3 s) was immediately
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delivered to the mice via the steel rods. After the electrical stimulation, the mice were
returned to their cages, and the apparatus was cleaned using a 10% ethanol solution to
remove residual odors. The time from when the door opened to when the mice enter the dark
compartment was recorded in seconds as the step-through latency. One hour before the
acquisition trial, mice in the Control, Vehicle and DON 4 groups were administered saline,
saline or donepezil 4mg·kg-1, respectively (p.o.). Except for those in Control group, all mice
were administered 1 mg·kg-1 scopolamine (i.p.) after 30 min. Mice in the KJH-1002-10 and
KJH-1002-20 groups were administered 10 or 20 mg·kg-1 KJH-1002 (p.o.), respectively, 25
min after the scopolamine injection. Thirty minutes after the scopolamine injection, the mice
were allowed to explore in the passive avoidance task. Twenty-four hours after the
acquisition trial, the retention trial was carried out to measure the long-term avoidance
memory. The mice were placed in the illuminated compartment, the guillotine door was
opened after 30 s, and the mice were then allowed to freely enter the dark compartment. The
step-through latencies were recorded with a maximum of time 180 s and used as an indicator
of avoidance memory.
Novel Object Recognition (NOR) Test
The NOR test is designed based on whether rodents are more interested in novel rather than
familiar objects (Francis et al., 2012). The test was conducted during four consecutive days,
divided into three phases: habituation (days one and two), introduction (day three) and
recognition (day four). Briefly, a gray, polyvinyl plastic open-field arena (50 x 50 x 50 cm)
was fixed in a soundproof room. During the habituation phase, mice were placed in the arena
in the absence of any objects and allowed to explore the environment for 5 min each day.
During the introductory phase, mice were placed in the arena for an exploration period in the
absence of any object. After 2 min, two identical objects (the same in texture, color and size)
were placed in two opposite corners of the arena 8 cm from the wall. The mice were allowed
to explore the objects for 3 min. One hour before the NOR test, mice in the Control, Vehicle
and DON groups were administered saline, saline or donepezil 4mg·kg-1, respectively (p.o.).
Except for those in the Control group, all mice were administered 1 mg·kg-1 scopolamine (i.p.)
after 30 min. Mice in the KJH-1002-10 and KJH-1002-20 groups were administered 10 or 20
mg·kg-1 KJH-1002 (p.o.), respectively, 25 min after the scopolamine injection. The
introductory phase trial was carried out 30 min after the scopolamine administration. Twenty-
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four hours after the introductory phase, one of the two old identical objects was replaced by a
novel object (different in texture, color and size) during the recognition phase. Exploratory
behavior was recorded when a mouse sniffed or touched the objects with its nose or paws.
Time spent exploring the objects (both old and novel objects) was recorded using a stopwatch.
To remove the residual odors, a 10% ethanol solution was used to clean the arena and the
objects after each mouse. A discrimination index was calculated by the following formula: DI
= [Tn / (Tn + Tf)] x 100 where Tn is the time spent exploring the novel object and Tf is the
time spent exploring the familiar object.
Tissue preparation
The day after the behavioral tests, mice in the Control, Vehicle and DON 4 groups were
administered saline, saline or 4mg·kg-1 donepezil, respectively (p.o.), and all mice except for
those in the Control group were administered 1 mg·kg-1 scopolamine (i.p.) after 30 min. Mice
in the KJH-1002-10 and KJH-1002-20 groups were administered 10 or 20 mg·kg-1 KJH-1002
(p.o.), respectively, 25 min after the scopolamine injection. Thirty minutes after the
scopolamine administration, mice were sacrificed by cervical dislocation. The skulls were
dissected, and the brain cortices and hippocampi were carefully collected and stored at -80°C
before use. Next, the brain tissues (part of the cortex and all of the hippocampus) were
homogenized in PRO-PREPTM lysis buffer (iNtRON, Gyeonggi, Korea) supplemented with
Phosphatase Inhibitor Cocktail Set I (Sigma-Aldrich, MO, USA). The homogenates were
stored at 4°C for 30 min and then centrifuged at 13000xg at 4°C for 30 min. The supernatants
were collected, and the protein concentrations were assayed by the Bradford method with a
standard curve constructed using BSA. A portion of the supernatants was diluted by adding
loading buffer to a concentration of 1.5 mg·mL-1 and then denatured by heating at 99°C for 5
min. The denatured and non-denatured parts of the supernatants were stored at -80°C for
future western blot and enzyme activity analyses.
Determination of cGMP level in the cortex
An immunoassay kit (BioVision, Milpitas, CA, USA) was used to analyze the cGMP levels in
the cortex, as reported by (Fiorito et al., 2013). Other part of the cortex was weighted and
homogenized with 5 volumes of 0.1 M HCl, the homogenate was centrifuged at 13000 x g for
5 min and the supernatant was collected. After the protein concentration was determined, the
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supernatants were used in the immunoassay, which was conducted according to the
manufacturer’s instructions. Each sample was assayed in duplicate, and the OD450 intensity
was inversely proportional to the concentration of cGMP, which is expressed as pmol·mg-1
protein.
Western blot analysis
To measure the protein marker expression levels in the cortex and hippocampus, the brain
samples (20 µg) were separated using 12% or 8% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). The proteins were then transferred onto polyvinylidene fluoride
(PVDF) membranes using a Trans-Blot Turbo transfer system (Bio-Rad, USA) in transfer
buffer. After transfer, the membranes were further incubated in blocking buffer (5% skim
milk in tris-buffered saline with 0.1% Tween 20) for 1 h at room temperature and then
incubated in rabbit monoclonal primary antibodies (anti-BDNF antibody, anti-p-CREB
antibody, anti-CREB, anti-p-ERK, anti-ERK, anti-p-Akt, anti-Akt and anti-GAPDH diluted
in blocking buffer at a ratio of 1:1000) at 4°C overnight. After incubation with the primary
antibodies, the membranes were washed three times with wash buffer (tris-buffered saline
with 0.1% Tween 20, TBST) for 30 min, and then incubated with horseradish peroxidase-
conjugated goat anti-rabbit secondary antibodies (ratio of 1:3000) in blocking buffer for 1 h
at room temperature. After being washed three times for 30 min, the protein bands were
developed using an ECL detection kit according to standard procedure and visualized by the
LAS-4000 mini system (Fujifilm, Japan). The protein band intensities of were analyzed using
Multi Gauge V3.0 software (Fujifilm, Japan) and normalized according to the GAPDH
intensity.
Analysis of AChE activity
AChE activity in the cortex and hippocampus were measured using the protocol described by
Ellman et al. (1961). In brief, 40 µL of PBS (50 mM, pH 7.0) and 10 µL of supernatants (1.5
mg·kg-1) were added to the wells of a 96-well cell culture plate and mixed by gentle shaking.
The reaction was initialized when a 50 µL mixture of Acetylthiocholine iodide (AChI, 10
mM in 50 mM PBS, pH 7.0) and 5, 5´-dithiobis (2-nitrobenzoic acid) (DTNB, 10 mM in 50
mM PBS, pH 8.0) was added. The absorbance at 412 nm was immediately measured using a
microplate spectrophotometer (BioTek, VT, USA) with a kinetic mode (at an interval of 1
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min) for 20 min. The absorbance value at 5 min and a molar extinction coefficient of 14.15
mM-1cm-1 was employed to calculate the final AChE activity, which is expressed as
nmol·min-1· mg-1 protein.
Measurement of the MDA levels
Malondialdehyde (MDA) is a product of lipid peroxidation, and the MDA levels in the cortex
and hippocampus were measured by the method provided by Esterbauer et al. (1990) with
slight modifications. Briefly, 240 µL of freshly prepared 0.5% phosphoric acid (pH 2.0), 80
µL of 0.6% tiobarbituric acid and 40 µL of prepared cortex or hippocampus homogenates
were mixed in a 1.5 mL micro-centrifuge tube. The mixture was heated at 95°C for 1 h. and
after cooling, an additional 300 µL of n-butanol was added and shaken vigorously. The
mixture was centrifuged at 13000 x g for 5 min, 200 µL of the n-butanol layer (pink color)
was collected, and the absorbance at 532 nm was measured using a microplate
spectrophotometer (BioTek, VT, USA) with an endpoint mode. A molar extinction coefficient
of 156 mM-1cm-1 was used to calculate the MDA level, which is expressed as nmol·mg-1
protein.
Measurement of SOD activity
Measurement of superoxide dismutase (SOD) activity was mainly performed according to the
method described by Marklund et al. (1974) with minor modifications. The method was
based on the inhibitory effects of SOD on the autoxidation of pyrogallol in alkaline solution.
Briefly, 700 µL of tris-cacodylic solution (pH 8.5) containing 20 mM
ethylenediaminetetraacetic acid (EDTA, St. Louis, MO, USA), 20 mM
diethylenetriaminepentaacetic acid (DTPA, St. Louis, MO, USA) and 200 µg·mL-1 catalase
was mixed with 200µL of the cortex or hippocampus supernatant in the well of 48-well cell
culture plate. Autoxidation was initiated by adding 100 µL of 3 mM freshly prepared
pyrogallol (in 10 mM HCl), the absorbance at 420 nm was recorded from 1.5 min to 3.5 min
with in a kinetic mode (interval of 30 s). A standard SOD substance (1 mg equal to 5763
units, St. Louis, MO, USA) was employed to construct a standard curve. SOD activity in the
cortex or the hippocampus was read according to the standard curve and is expressed as
U·mg-1 protein.
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Estimation of GPx activity
Quantitative analysis of glutathione peroxidase (GPx) activity in the cortex and hippocampus
was carried out according to the method described by Ellman (1959) and Hancianu et al.
(2013) with slight modifications. Briefly, 20 µL of brain tissue homogenate was mixed with
20 µL of 2 mM glutathione (GSH) in PBS solution (0.2 M, pH 7.0) containing 2.5 mM NaN3
and 0.2 mM EDTA. After incubating at 37°C for 5 min, 10 µL of 0.6 mM H2O2 was added
and incubated at 37°C for 5 min. Then an additional 100 µL of 5% trichloroacetic acid (TCA
in ultrapure water) was added and incubated for 10 min at room temperature. After
centrifugation at 3000 x g for 10 min, the reaction was initiated by mixing 100 µL of
supernatant and 35 µL of 4% DTNB (in ultrapure water). Absorbance of the yellow color was
immediately measured at 412 nm by a microplate spectrophotometer (BioTek, VT, USA) with
an endpoint mode. A reaction system in which the homogenate was replaced with PBS
solution (0.2 M, pH 7.0) served as a control. The GPx activity was calculated using a molar
extinction coefficient of 14.15 mM-1cm-1 and expressed as nmol·min-1·mg-1 protein.
Estimation of GR activity
We determined the glutathione reductase (GR) activity in the cortex and hippocampus
according to the method provided by Ellman (1959). The reaction system contained 20 µL of
assay buffer (0.2 M PBS containing 2.5 mM NaN3 and 0.2 mM EDTA pH 7.0), 100 µL of
oxidized glutathione (GSSG, 2 mM in assay buffer), 20 µL of brain tissue homogenate and 50
of µL DTNB (3 mM in assay buffer). The reaction was immediately initiated when 10 µL of
β-nicotinamide adenine dinucleotide 2´-phosphate reduced tetrasodium salt hydrate
(NADPH, 2 mM in assay buffer) was added. After 5 min, the absorbance at 412 nm was
measured by microplate spectrophotometer. A molar extinction coefficient of 14.15 mM-1cm-1
was used to calculate the GR activity, which is expressed as nmol·min-1·mg-1 protein.
Statistical analysis
All data were analyzed using GraphPad Prism 7 software (GraphPad Software, CA, USA)
and are presented as mean ± SEM. The escape latency data from MWM test were analyzed
by two-way analysis of variance (ANOVA) followed by the post hoc Tukey’s multiple
comparison tests. All other data were analyzed by one-way ANOVA followed by the post hoc
Dunnett’s multiple comparison tests. Post hoc tests were run only when F value achieved P <
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0.05. Statistical significance was considered when a value of P < 0.05 was confirmed. The
operator and data analysis were blinded. The data and statistical analysis comply with the
recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015).
Materials
Scopolamine hydrobromide, donepezil, 5,5´-Dithiobis(2-nitrobenzoic acid), acetylthiocholine
iodide, thiobarbituric acid, phosphoric acid, pyrogallol, catalase, superoxide dismutase,
glutathione, L-glutathione oxidized, nicotinamide adenine dinucleotide phosphate and anti-
BDNF antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-CREB,
anti-phosphorylated CREB (p-CREB), anti-ERK, anti-phosphorylated ERK (p-ERK), anti-
Akt, anti-phosphorylated Akt (p-Akt) and anti-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) antibodies were purchased from Cell Signaling Technology (MA, USA). All other
reagents were of the highest grade and were purchased from commercial resources.
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).
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Results
Effect of KJH-1002 on inhibiting PDE5
The KJH-1002 compound exhibited excellent inhibitory activity (Table 1) against PDE5 with
an IC50 value of 0.059  0.04 nmol·L-1, and it also showed inhibitory activities against PDE1
(IC50 = 2.03  0.59 mol·L-1), PDE3 (IC50 = 1.17  0.99 mol·L-1), PDE4 (IC50 = 920.12 
68.17 nmol·L-1) and PDE6 (IC50 = 47.44  27.79 nmol·L-1). Based on these data, KJH-1002
can be considered as a specific PDE5 inhibitor. Meanwhile, due to the limitation of the test,
we can’t rule out the possibility that KJH-1002 exhibits stronger inhibitory activity against
other subtypes of PDE.
Effects of KJH-1002 on spatial learning and memory in the MWM test
The effect of KJH-1002 on scopolamine-impaired long-term spatial learning and memory
was assessed by the MWM test. As shown in Figure 2A, mice in the control group rapidly
learned and memorized the location of the platform in the target quadrant and exhibited the
shortest escape latencies during the acquisition phase. The scopolamine-treated (1 mg·kg-1)
group showed longer average escape latency, and significant differences were observed
compared with the control group on day 4 and day 5, indicating that an amnesia model was
successfully induced by scopolamine injection. KJH-1002 (20 mg·kg-1) and donepezil (4
mg·kg-1) significantly decreased the scopolamine-prolonged escape latency from day 4
onward. The day after the acquisition phase, all mice were subjected to a probe trial by
removing the platform. Mice in the scopolamine-treated group spent a markedly shorter time
swimming in the target quadrant than mice in the control group (Figure 2B). The shortened
swimming time in the target quadrant was increased by donepezil or KJH-1002
administration.
Effect of KJH-1002 on scopolamine-impaired spontaneous alternation behavior in the
Y-maze test
The effect of KJH-1002 on scopolamine-impaired short-term spatial cognition was evaluated
using the Y-maze test. The ratio of spontaneous alternation behavior in the Y-maze test was
used as an important index to assess the learning and memory abilities of the mice.
Scopolamine injection resulted in significantly decreased ratio of spontaneous alternation
behavior compared with that of the control group (Figure 2D), and the scopolamine-
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decreased alternation behavior was significantly recovered by administration of donepezil or
KJH-1002 (10 and 20 mg·kg-1). Moreover, scopolamine injection also resulted in significant
increase in the number of arm entries in comparison with control group (Figure 2E).
Interestingly, KJH-1002 (20 mg·kg-1) dramatically reversed the scopolamine-increased
number of arm entries, indicating improved locomotor activity.
Effect of KJH-1002 on step-through latency in the passive avoidance test
The passive avoidance test was conducted to assess the enhancing effect of KJH-1002 on
scopolamine-impaired step-through latency. As shown in Figure 2C, no significant
differences in step-through latency were found among any of the groups in the acquisition
trial. However, in the retention trial, a significant decrease in step-through latency was
induced by scopolamine administration compared with that of the control group, which
revealing that long-term memory impairment was induced by scopolamine. Interestingly, the
decrease in step-through latency was significantly improved by KJH-1002 (20 mg·kg-1) or
donepezil (4 mg·kg-1).
Effect of KJH-1002 on scopolamine-induced memory impairment in the NOR test
Recognition memory was evaluated using the NOR test in which the time mice spent
exploring novel and familiar objects was monitored, and object recognition memory was
assessed by a defined discrimination index. As shown in Figure 2F, mice in the control group
clearly preferred the novel object more than the familiar one, while scopolamine significantly
reduced the time mice spent exploring the novel object, as a dramatic decline in the
discrimination index was observed. Pretreatment with donepezil or KJH-1002 (at both 10 and
20 mg·kg-1) significantly improved the discrimination index, demonstrating that
scopolamine-impaired recognition memory was reversed by KJH-1002 administration.
The effect of KJH-1002 on cGMP levels in the cortex
The effect of KJH-1002 on the cGMP level in the cortex was measured using a direct
competitive immunoassay kit. As shown in Figure 3, scopolamine administration failed to
impact the cGMP levels in the cortex compared with those in the control group. Similarly, no
significant increase in cGMP levels was found in the donepezil-treated group in comparison
with the scopolamine or control groups. Interestingly, KJH-1002 markedly increased the
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cGMP levels in the cortex, and the high dose 20 mg·kg-1 dose especially exhibited a
significant effect on increasing the cGMP levels.
Effect of KJH-1002 on the expression of memory-related protein molecules in the cortex
and hippocampus
To elucidate the molecular mechanism underlying how KJH-1002 exerts cognition-
enhancing effects, we investigated the phosphorylation levels of CREB, ERK1/2 and Akt, as
well as BDNF expression in the cortex and hippocampus. Figure 4A shows the representative
immunoblots in the cortex, and Figure 4C shows that scopolamine significantly reduced the
cortical phosphorylation levels of CREB, ERK1/2 and Akt, and the BDNF level compared
with those in the control group. Pretreatment with donepezil, the positive control,
significantly reversed the phosphorylation levels of CREB and Akt, as well as BDNF
expression in the cortex. KJH-1002 (20 mg·kg-1) administration significantly enhanced the
phosphorylation of CREB, ERK, and the expression of BDNF in the cortex. Interestingly,
KJH-1002 at doses of 10 and 20 mg·kg-1 significantly enhanced the cortical phosphorylation
levels of Akt. Similarly, Figure 4B and Figure 4D shows that the phosphorylation levels of
CREB, ERK1/2 and Akt, as well as the expression of BDNF in the hippocampus were
significantly decreased by scopolamine administration in comparison with the control group.
The decreased phosphorylation of CREB, ERK1/2 and Akt, as well as the expression of
BDNF were significantly reversed by donepezil administration. Both 10 and 20 mg·kg-1 of
KJH-1002 significantly reversed the phosphorylation levels of CREB and ERK1/2 in the
hippocampus. Significant enhancing effects on Akt phosphorylation and BDNF expression in
the hippocampus were observed in the group pretreated with 20 mg·kg-1 KJH-1002.
Effect of KJH-1002 on inhibiting the activity of AChE
Scopolamine is competitive muscarinic receptor antagonist, which can block the
neurotransmitter acetylcholine from binding the muscarinic receptor. To investigate whether
KJH-1002 exerted its cognition-enhancing effects by inhibiting AChE activity, we measured
the AChE activity in the brain tissues. The cortical and hippocampal AChE activities were
significantly increased by scopolamine administration in comparison with the activity in the
control group (Figure 5), and the scopolamine-increased AChE activities in the cortex and
hippocampus were dramatically inhibited by donepezil administration. However, KJH-1002
administration failed to reduce the AChE activity increased by scopolamine injection in the
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cortex or hippocampus, suggesting that KJH-1002 did not exert its protective effect on
cognitive function by inhibiting AChE activity.
Effect of KJH-1002 on anti-oxidative stress
Lovell et al. (1995) and Markesbery (1997) emphasized that increased oxidative stress is
clearly involved in neuron degeneration and death in AD. Our research examined the levels
of MDA, an important marker of lipid peroxidation, and the activity levels of superoxide
dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) in the cortex
and hippocampus. The cortical and hippocampal MDA levels were significantly increased by
scopolamine administration compared with the levels in the control group (Figure 6A).
Administration of donepezil (4 mg·kg-1) and KJH-1002 at doses of 10 and 20 mg·kg-1
significantly decreased the MDA level in the cortex. In the hippocampus, 4 mg·kg-1 donepezil
and 20 mg·kg-1 KJH-1002 significantly affected the decreased MDA level induced by
scopolamine.
Figure 6B shows changes in SOD activity, and significantly decreased SOD activity
was induced by scopolamine compared with the levels in the control group in both the
cortex and hippocampus. Additionally, decreased SOD activity was significantly restored by
administration of 4 mg·kg-1 donepezil or KJH-1002 at doses of 10 and 20 mg·kg-1.
Figure 6C shows that the GPx activity in the cortex and hippocampus was significantly
decreased by scopolamine compared with the activity in the control group. The scopolamine
induced decrease in GPx activity was significantly reversed by administration of donepezil (4
mg·kg-1) or KJH-1002 (10 and 20 mg·kg-1) in the cortex and hippocampus.
The effects of scopolamine and KJH-1002 on the GR activity in cortex and hippocampus
are shown in Figure 6D. Significant decreases in cortical and hippocampal GR activity levels
were found in the scopolamine-treated group compared with the levels in the control group.
Donepezil significantly enhanced the GR activity reduced by scopolamine in both the cortex
and the hippocampus. Moreover, the high dose of KJH-1002 (20 mg·kg-1) resulted in
significantly increased GR activity in the cortex, while KJH-1002 at doses of 10 and 20
mg·kg-1 significantly increased the GR activity in the hippocampus compared with the
activity in the scopolamine-treated group.
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Discussion
The cGMP specific hydrolyzer, PDE5 was pointed out as a potential target for the treatment
of neurologic diseases (Garcia-Osta et al., 2012). Teich et al. (2016) reported the presence of
PDE5 in human brain. In the present study, we synthesized an analogue of sildenafil, KJH-
1002, that shows potent inhibition against PDE5 (IC50 = 0.059 ± 0.04 nmol·L-1). In addition,
KJH-1002 also exhibits inhibitory activity against PDE1, PDE3, PDE4 and PDE6. Because
the PDE5 inhibitor sildenafil has been confirmed to enhance cognitive function in a variety of
animal models, we tested the cognition-enhancing effect of KJH-1002 on a scopolamine-
induced amnesia mouse model. The results revealed that KJH-1002 could reverse various
kinds of learning and memory in a series of behavioral tests. Biochemical analysis indicated
that KJH-1002 may improve learning and memory by activating the cGMP/CREB signaling
pathway and inhibiting oxidative stress damage without influencing AChE activity. PDE2
hydrolyzes both cAMP and cGMP, PDE9 is a cGMP-specific hydrolyzer, and inhibition of
these two subtypes of PDE leads to the accumulation of cGMP and improves the memory
performance in a variety of animal model (Boess et al., 2004; Kleiman et al., 2012). Due to
the limitation of the present study, the inhibitory activity of KJH-1002 against PDE2 and
PDE9 remains unknown. We can’t rule out the possibility that beneficial effects of KJH-1002
on scopolamine impaired animal model resulting from its inhibitory activity against these two
PDE subtypes.
Scopolamine is a standard drug that can impair cognitive function in healthy humans
and animals via disturbing the neurotransmitter acetylcholine. Administration of scopolamine
affects a variety of learning and memory, and locomotor activity, thus induces abnormal
behavioral processes (Klinkenberg et al., 2010). After pretreatment with KJH-1002, all mice
were subjected to the MWM test. Scopolamine dramatically increased the escape latency for
finding the hidden platform, and 20 mg·kg-1 KJH-1002 and 4 mg·kg-1 DON reversed the
spatial learning and memory impaired by scopolamine to an extent similar to that of the
normal control group. In supporting our finding, Cuadrado-Tejedor et al. (2011) reported a
cognition enhancing effect of sildenafil on an AD model of transgenic mouse in the MWM
test. Additionally, sildenafil was proved to attenuate scopolamine-induced learning and
memory impairment in a 14-unit T-maze test (Devan et al., 2004). By contrast, Prickaerts et
al. (2004) reported that a different PDE5 inhibitor, 10 mg·kg-1 zaprinast failed to enhance the
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performance of adult rats in MWM test. The difference may be attributed to the animal
model, as Prickaerts et al. (2004) showed an unimpaired rat model with optimal memory
performance, and it is difficult to enhance memory performance at a high level. While the
cognitive functions of our mouse model were significantly impaired by scopolamine, KJH-
1002 could rescue the cognitive functions from a low level. KJH-1002 may also exhibit some
inhibitory effects against phosphodiesterase 4 (IC50 = 920.12  68.17 nmol·L-1), a PDE4
inhibitor, as 3 mg·kg-1 rolipram reportedly improved learning and memory in a microsphere
embolism-induced cerebral ischemia rat in MWM test (Nagakura et al., 2002). The Y-maze
test is used to assess short-term memory by analyzing spontaneous alternation behavior
(Kwon SH et al., 2013). Scopolamine administration significantly damaged the spontaneous
alternation behavior compared with that of the control group. KJH-1002 at doses 10 and 20
mg·kg-1 and donepezil at a dose of 4 mg·kg-1 could ameliorate the scopolamine-impaired
spontaneous alternation behavior back to the normal level. Moreover, KJH-1002 decreased
the number of arm entries increased by scopolamine, revealing that KJH-1002 reduces the
scopolamine-induced increase of locomotor activity (Klinkenberg et al., 2010). Numerous
studies have shown that PDE5 inhibitors can enhance long-term memory in the passive
avoidance test. Sildenafil was reported to enhance memory in unimpaired young and aged
mouse (Patil et al., 2004), as well as reverse memory performance in streptozotocin-induced
diabetic rats and electroconvulsive shock-stimulated rats in the passive avoidance test (Patil
et al., 2006). Our study shows that the PDE5 inhibitor KJH-1002 also plays an important
protective role against scopolamine-induced cognitive dysfunction in passive avoidance.
Mice treated with KJH-1002 remembered the electric shock delivered during the acquisition
trial and exhibited longer step-through latency in the retention phase. This finding is
consistent with the studies by Van der Staay et al. (2008) showing that the inhibitor of
phosphodiesterase 9 (PDE9, also a cGMP-specific hydrolyzer), BAY 73-6691, ameliorated
long-term memory impairment induced by scopolamine in passive avoidance test. In the
NOR test of the present study, KJH-1002-treated mice preferred the novel object, as they
spent more time exploring the new object compared to the mice administered only
scopolamine demonstrating that KJH-1002 also improves the object recognition memory
deficit induced by scopolamine. This finding is consistent with results revealed by van der
Staay et al. (2008) and Hutson et al. (2011), who found that the PDE9 inhibitors, BAY 73-
6691 and PF-04447943 significantly enhanced the memory performance in unimpaired and
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scopolamine impaired rats in NOR test, respectively.
Scopolamine is well known to impair learning and memory in various types of behavior
tests by not only blocking the cholinergic system, but also reducing CREB and BDNF
expression (Shi et al., 2013; Hong et al., 2014; Jeon et al., 2017). The transcription factor
CREB serves as a molecular switch in learning and memory. CREB Phosphorylation plays an
important role in activating transcription, thus leads to the expression of downstream genes,
such as BDNF, which is an important protein for synaptic plasticity and memory performance
(Huang et al., 2001; Scott Bitner, 2012). Several studies have suggested that CREB and
BDNF have therapeutic potential in AD patients (Bejar et al., 1999; Nagahara et al., 2009).
PDE5 inhibition activates CREB, leading to transcriptional promotion of the target BDNF
gene and thereby promoting synaptic plasticity and memory formation (Cuadrado-Tejedor et
al., 2011; Garcia-Osta et al., 2012). We found a significant decrease in p-CREB and BDNF
expression in the cortex and hippocampus after scopolamine administration, and subsequent
KJH-1002 administration could rescue expression of these proteins. Thus the enhancing
effect of KJH-1002 on cognitive function may be attributed to the recovery of p-CREB and
BDNF. Moreover, BDNF is the upstream molecular effector of phosphorylated ERK 1/2
responsible for establishing synaptic activity and enhancing different types of memory
(Revest et al., 2014). Ota et al. (2008) suggested an involvement of the lateral amygdala
ERK/MAPK in the NO-cGMP-PKG signaling pathway in modulating memory consolidation.
We observed that KJH-1002 could also significantly reverse the cortical and hippocampal
expression of p-ERK after the recovery of p-CREB and BDNF, which suggests an
involvement of activated ERK in the promotion of cognition function. Akt, an enhancer of
neuronal survival, serves as a signal transducer of neurotrophin-regulated survival and is
important for the prevention of various neurodegenerative challenges (Calabrese et al., 2007).
Interestingly, we found that KJH-1002 counteracted the effect of scopolamine and induced a
dramatic increase in Akt phosphorylation supporting an important role of Akt in reducing
scopolamine induced cognitive dysfunction. Western blot analysis showed that activation of
the CREB signaling pathway by KJH-1002 plays a key role in reversing cognitive
impairments induced by scopolamine.
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Several studies have suggested that PDE5 inhibitors elevate cGMP levels in various
brain regions, such as the cortex, hippocampus and cerebellum, in transgenic AD mice or
unimpaired rats, and cGMP elevation promotes CREB phosphorylation, thus improving
memory performance in different types of behavior tests (Marte et al., 2008; Puzzo et al.,
2009; Jin et al., 2014). In this study, an enzyme-linked immunosorbent assay (ELISA) kit was
used to determine cGMP level in the cortex. Scopolamine and donepezil did not affect cGMP
levels in the cortex, while the PDE5 inhibitor KJH-1002 increased the cortical cGMP levels
to a dramatically high level. Thus, KJH-1002 ameliorated cognitive dysfunction is dependent
upon the enhancement of cGMP level.
Scopolamine-induced amnesia is also characterized by the increased activity of AChE,
which interferes with learning and memory by catalyzing the neurotransmitter acetylcholine
into choline and acid (Gautam et al., 2016; Kulshreshtha et al., 2016; Nedaei et al., 2016). In
the present study, KJH-1002 did not inhibit the increased AChE activity caused by
scopolamine, indicating that cognitive function enhancement by KJH-1002 is not dependent
on AChE activity inhibition.
Overwhelming evidence has demonstrated that oxidative stress is involved in AD (Gella
et al., 2009). A study by Sikandaner et al. (2017) demonstrated that sildenafil administration
decreased oxidative stress and enhanced memory performance in mice impaired by chronic
noise exposure. In the present study, KJH-1002 exerted anti-oxidative effects on
scopolamine-impaired mice by significantly decreasing the MDA level and increasing
enzyme activities of SOD, GPx and GR in the cortex and hippocampus. Thus, a putative
effect of KJH-1002 on cognition enhancement is attributed to the potency of anti-oxidative
stress.
In conclusion, both the AChE inhibitor donepezil and the PDE5 inhibitor KJH-1002
ameliorate scopolamine-induced learning and memory decline. It is seemed that both of them
enhance cognitive function via modulating memory related proteins and attenuating oxidative
stress damage in a similar manner. The enhancing effect of donepezil on scopolamine
impaired cognitive function depends on the inhibition of AChE, while PDE5 inhibitor KJH-
1002 exerts the enhancing effect through increasing cGMP level.
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Acknowledgements
This work was funded and supported by the Bio-Synergy Research Project (NRF-
2012M3A9C4048793) and the Bio & Medical Technology Development Program (NRF-
2015M3A9A5030735) of the Ministry of Science, ICT, and Future Planning through the
National Research Foundation of the Republic of Korea to HOY. This work also was
supported by Korea Institute of Science and Technology (KIST) Institutional Program
(2E2685) and the Original Research Program (NRF-2016M3C7A1904344) funded by the
National Research Foundation, of Korea.
Author contributions
Ghilsoo Nam and Jae Hong Seo designed and synthesized the KJH-1002 compound. Hyun-
Ok Yang designed the in vivo experiments, and Lijun Zhang performed the animal behavior
tests. Huan Li analyzed the behavior results. Lijun Zhang performed the assays for
measurements of cGMP, MDA level, and enzymes activity, Huan Li analysis these results.
Huan Li carried out the western blot assay and Lijun Zhang analysis these results. Lijun
Zhang wrote the manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Declaration of transparency and scientific rigour
This Declaration acknowledges that this paper adheres to the principles for transparent
reporting and scientific rigour of preclinical research recommended by funding agencies,
publishers and other organisations engaged with supporting research.
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