Therapeutic evaluation of synthetic peucedanocoumarin III in an animal model of Parkinson’s disease

Article abstract of DOI:10.3390/ijms20215481

The motor and nonmotor symptoms of Parkinson’s disease (PD) correlate with the formation and propagation of aberrant α-synuclein aggregation. This protein accumulation is a pathological hallmark of the disease. Our group recently showed that peucedanocoumarin III (PCIII) possesses the ability to disaggregate β sheet aggregate structures, including α-synuclein fibrils. This finding suggests that PCIII could be a therapeutic lead compound in PD treatment. However, the translational value of PCIII and its safety information have never been explored in relevant animal models of PD. Therefore, we first designed and validated a sequence of chemical reactions for the large scale organic synthesis of pure PCIII in a racemic mixture. The synthetic PCIII racemate facilitated clearance of repeated β sheet aggregate (β23), and prevented β23-induced cell toxicity to a similar extent to that of purified PCIII. Given these properties, the synthetic PCIII’s neuroprotective function was assessed in 6-hydroxydopamine (6-OHDA)-induced PD mouse models. The PCIII treatment (1 mg/kg/day) in a 6-OHDA-induced PD mouse model markedly suppressed Lewy-like inclusions and prevented dopaminergic neuron loss. To evaluate the safety profiles of PCIII, high dose PCIII (10 mg/kg/day) was administered intraperitoneally to two-month-old mice. Following 7 days of PCIII treatment, PCIII distributed to various tissues, with substantial penetration into brains. The mice that were treated with high dose PCIII had no structural abnormalities in the major organs or neuroinflammation. In addition, high dose PCIII (10 mg/kg/day) in mice had no adverse impact on motor function. These findings suggest that PCIII has a relatively high therapeutic index. Given the favorable safety features of PCIII and neuroprotective function in the PD mouse model, it may become a promising disease-modifying therapy in PD to regulate pathogenic α-synuclein aggregation.

Full text of DOI:10.3390/ijms20215481

International Journal of
Molecular Sciences
Article
Therapeutic Evaluation of Synthetic
Peucedanocoumarin III in an Animal Model of
Parkinson’s Disease
Sangwoo Ham 1,†,‡, Heejeong Kim 1,‡, Jin-Ha Yoon 2,‡, Hyojung Kim 1, Bo Reum Song 3,
Jeong-Yun Choi 1, Yun-Song Lee 1, Seung-Mann Paek 3,*, Han-Joo Maeng 2,* and
Yunjong Lee 1,
*
1
Division of Pharmacology, Department of Molecular Cell Biology, Sungkyunkwan University School of
Medicine, Samsung Biomedical Research Institute, Suwon 16419, Korea; ham89p12@skku.edu (S.H.);
kimhj9301@gmail.com (H.K.); hjung93@skku.edu (H.K.); choijy@skku.edu (J.-Y.C.); yslee@skku.edu (Y.-S.L.)
College of Pharmacy, Gachon University, Incheon 21936, Korea; jinha89@daum.net
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University,
Jinju Daero 501, Jinju 52828, Gyeongnam, Korea; qhfma2005@naver.com
2
3
*
†
‡
Correspondence: million@gnu.ac.kr (S.-M.P.); hjmaeng@gachon.ac.kr (H.-J.M.); ylee69@skku.edu (Y.L.)
Present address: ToolGen Inc., Gasan Digital-Ro, Geumcheon, 08501, Seoul, Korea.
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 October 2019; Accepted: 1 November 2019; Published: 4 November 2019
Abstract: The motor and nonmotor symptoms of Parkinson’s disease (PD) correlate with the formation
and propagation of aberrant
hallmark of the disease. Our group recently showed that peucedanocoumarin III (PCIII) possesses
the ability to disaggregate sheet aggregate structures, including -synuclein fibrils. This finding
α-synuclein aggregation. This protein accumulation is a pathological
β
α
suggests that PCIII could be a therapeutic lead compound in PD treatment. However, the translational
value of PCIII and its safety information have never been explored in relevant animal models of PD.
Therefore, we first designed and validated a sequence of chemical reactions for the large scale organic
synthesis of pure PCIII in a racemic mixture. The synthetic PCIII racemate facilitated clearance of
repeated β sheet aggregate (β23), and prevented β23-induced cell toxicity to a similar extent to that
of purified PCIII. Given these properties, the synthetic PCIII’s neuroprotective function was assessed
in 6-hydroxydopamine (6-OHDA)-induced PD mouse models. The PCIII treatment (1 mg/kg/day)
in a 6-OHDA-induced PD mouse model markedly suppressed Lewy-like inclusions and prevented
dopaminergic neuron loss. To evaluate the safety profiles of PCIII, high dose PCIII (10 mg/kg/day) was
administered intraperitoneally to two-month-old mice. Following 7 days of PCIII treatment, PCIII
distributed to various tissues, with substantial penetration into brains. The mice that were treated
with high dose PCIII had no structural abnormalities in the major organs or neuroinflammation.
In addition, high dose PCIII (10 mg/kg/day) in mice had no adverse impact on motor function.
These findings suggest that PCIII has a relatively high therapeutic index. Given the favorable safety
features of PCIII and neuroprotective function in the PD mouse model, it may become a promising
disease-modifying therapy in PD to regulate pathogenic α-synuclein aggregation.
Keywords: peucedanocoumarin III; organic synthesis; Parkinson’s disease; distribution; α-synuclein
aggregation; dopaminergic cell loss
1. Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder of which
characteristic pathological features include Lewy bodies, intracellular inclusions that are mainly
Int. J. Mol. Sci. 2019, 20, 5481; doi:10.3390/ijms20215481
www.mdpi.com/journal/ijms

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composed of misfolded and phosphorylated
α
-synuclein [
1]. Pathological Lewy bodies in PD are
first found in the lower brain stem, after which they spread to the midbrain and finally to the
cortex in the advanced stages [ ]. This pathological a-synuclein aggregation in diverse brain
subregions corresponds to the progression of clinical motor and nonmotor symptoms in PD [ ].
2–4
2–4
Currently available treatments for the motor and non-motor symptoms of PD address only symptoms.
In particular, L-DOPA supplements increase dopamine neurotransmission, and therefore control
the clinical motor deficits in PD. However, the therapeutic effects of L-DOPA decline as the disease
progresses. This treatment eventually results in adverse effects such as dyskinesia. Therefore, it is
imperative to develop disease-modifying therapies that halt or reverse the ongoing pathological
propagation of
α
-synuclein pathologies and neurodegeneration. Specifically, therapeutic strategies to
-synuclein propagation
regulate Lewy body inclusion may hold promise in controlling pathological
α
in various brains subregions. These strategies may have more therapeutic value in treating the motor
and nonmotor symptoms that are caused by Lewy body pathology.
Several compounds have been reported to have disaggregating ability against fibrillar
in in vitro or cell systems. However, only a few compounds have been evaluated in animal models of
PD. For instance, Nilotinib, the brain permeable c-Abl inhibitor, has been shown to reduce -synuclein
aggregation and dopaminergic neurodegeneration in PD mouse models [ ]. In addition, the stable
GLP-1 agonist was also shown to control -synuclein pathologies via regulation of microglia and
α-synuclein
α
5,6
α
astrocyte conversion [7]. In addition to these therapeutic evaluations in PD mouse models, extensive
safety profiling of potential lead compounds are also important. This is particularly true for translational
research aiming for clinical trials in the future. In order to control for the potential toxicity of prolonged
therapies in neurodegenerative disorders, it is essential to understand the potential alterations in organ
integrities or behavior in mice with the treatment.
Our group previously identified that peucedanocoumarin III (PCIII) has strong inhibitory
properties against artificially repeated
α
β
sheet aggregate toxicity [
-synuclein fibrils in vitro and prevented -synuclein induced toxicity in cells. In this case, PCIII
] was used. Despite PCIII’s therapeutic potential for PD, translational
8]. In addition, PCIII disaggregated
α
purified from A. decursiva roots [
8
research has not been attempted because of issues with stable-and large-scale production of qualitatively
consistent PCIII. In this study, we developed an organic synthesis protocol for highly pure PCIII
production. We demonstrated therapeutic functions of PCIII in the regulation of α-synuclein aggregation
and dopamine cell viability in a PD mouse model. Importantly, we also thoroughly evaluated the
tissue distribution and safety information of synthetic PCIII in vivo.
2. Results
2.1. Organic Synthesis of Peucedanocoumarin III and its Evaluation
To ensure consistent and reliable therapeutic outcomes with in vivo PCIII treatment, it is essential
to standardize the large scale organic synthesis of highly pure PCIII. We designed chemical reaction
procedures for the organic synthesis of PCIII. The detailed chemical synthesis of PCIII
Figure 1A. Umbelliferone was treated with acetal 3 followed by cyclization under thermal conditions to
produce good yield of seselin [ 11]. mCPBA epoxidation of alkene in seselin afforded corresponding
1 is outlined in
2
9–
4
racemic epoxide, which was transformed into trans-diol 5 through acidic hydrolysis [12,13]. We then
tried selective esterification of the trans-diol moiety. Some acylating agents such as tigloyl chloride or
acetic anhydride were esterified under weak basic conditions to provide the desired intermediate
its synthetic equivalents. The differentiation of two secondary alcohols, however, was not productive
after survey of the reaction conditions. Finally, the desired ester was obtained as a minor product
of tigloylation. The undesired isomer 14] was a major product. The standard acetylation protocol
then afforded PCIII as a racemate, which is identical to the authentic spectral data [ 14]. The PCIII
8 or
8
7
[
8
,
was produced in excellent yield. It was interesting that the acetylation/tigloylation sequence did not
produce PCIII because of its weak reactivity.

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Figure 1. Total synthesis of peucedanocoumarin III (PCIII) and evaluation of its anti-aggregate
effect. ( ) Scheme showing the detailed experimental steps in the organic synthesis of PCIII.
) Experimental schedule used to evaluate synthetic PCIII’s protective effect on Tet-Off-expressed 23
toxicity (top panel). Nuclear 23 was expressed for 24 h using the Tet-off system. Further expression
of 23 was halted by doxycycline (Dox) treatment. Cell viability was assessed 24 h after doxycycline
treatment. The degradation of 23 was monitored by Western blot (WB) and immunofluorescence
(IF) at 37 h after doxycycline treatment. The representative immunofluorescence images of FLAG
23) and the reporter protein mCherry in SH-SY5Y cells treated with the natural compound PCIII,
synthetic PCIII (1 M, 37 h), or DMSO as a vehicle. ( ) Quantification of relative nuclear FLAG ( 23)
immunofluorescence intensities in the reporter mCherry positive SH-SY5Y cells from the indicated
experimental groups (n = 21 cells from three independent experiments per groups). ( ) Representative
Western blot of nuclear β23 expression in SH-SY5Y cells treated with either natural or synthetic PCIII
or DMSO vehicle. 23 degradation was monitored using anti-FLAG antibodies. The control included
the SH-SY5Y cells transfected with pCMV-tTA and TetP mock vector without 23 expression. -actin
served as a loading control. ( ) Quantification of relative nuclear 23 protein levels normalized to
-actin for the indicated experimental groups (n = 3 per group). ( ) Cell viability assessed using
trypan blue exclusion assay (n = 6 per group), which showed the protective effect of both purified and
synthesized PCIII (1 µM each) in nuclear 23-expressing (48 h) SH-SY5Y cells. The control included the
SH-SY5Y cells, which were transfected with pCMV-tTA and TetP mock vector without 23 expression.
SEMs. *** p < 0.001, ANOVA test followed by Tukey post hoc
A
(B
β
β
β
β
(
β
µ
C
β
D
β
β
β
E
β
β
F
β
β
The data are expressed as means
analysis. NS, nonsignificant.
±
Since synthetic PCIII is produced as a racemate, we attempted to compare the biological functions
of synthetic PCIII with purified PCIII (that is extracted from natural herbs, as described previously) [ ].
To determine whether synthetic PCIII retains the ability to degrade artificial beta sheet structured
protein aggregate 23 [ 15], the SH-SY5Y cells were transiently transfected with both pCMV-tTA and
TetP-NLS-FLAG- 23-HA (TetP- 23) to produce doxycylcline-regulatable expression of aggregate 23
(Figure 1B, top panel). The steady state levels of 23 were monitored in the SH-SY5Y cells expressing
8
β
8,
β
β
β
β
reporter mCherry using immunofluorescence. This immunofluorescence was performed by labeling
the N terminal tag FLAG with the specific antibodies. In comparison to purified PCIII, the synthetic
PCIII treatment accelerated β23 clearance from the SH-SY5Y cells to a comparable extent (Figure 1B,C).

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We next sought to further confirm that the synthetic PCIII treatment degraded
β23. To do so we
exposed total protein lysates from the SH-SY5Y cells (pulsed 24 h expression of
blots using FLAG specific antibodies. The sustained presence of 23 in the DMSO vehicle group
was cleared up to approximately 60% with the purified and synthetic PCIII treatment (Figure 1D,E).
Synthetic PCIII provided potent cytoprotection against 23 in a dose-dependent manner, with maximal
protective effect at 1 M concentration (Supplementary Figure S1A). In addition, consistent with the
degradation of 23, 23-induced ~80% cell death in DMSO control group was reduced to around
β23) to Western
β
β
µ
β
β
40% cell death after treatment of either purified or synthetic PCIII (Figure 1F). We also examined
PCIII’s cytoprotective function in another PD-related cellular model, 6-OHDA-intoxicated SH-SY5Y
cell that has been reported to produce
α-synuclein aggregation and cytotoxicity [8,16]. Following
HA- -synuclein transient transfection to SH-SY5Y cells, subsequent 6-OHDA treatment led to robust
α
cell toxicity of about 80% cell death (Supplementary Figure S1B). Synthetic PCIII treatment provided
partial cytoprotection in this PD-related cell model (Supplementary Figure S1B). These results suggest
that synthetic PCIII has similar biologic activity to that of purified PCIII, including β23 degradation
and cytoprotection against β23 and 6-OHDA-induced toxicity.
2.2. PCIII Prevents DA Cell Loss and α-synuclein Aggregation in a 6-OHDA PD Mouse Model
We previously reported that PCIII treatment can prevent oxidative stress-induced α-synuclein
aggregation and toxicity in cell systems [
an intrastriatal injeciton of 6-OHDA as a mouse model of PD (Figure 2A). The 6-OHDA-induced
PD mouse model has been widely used to model dopaminergic neuronal degeneration [17 18] and
-synuclein aggregation [16]. To evaluate the protective function of synthetic PCIII, PCIII was
administered intraperitoneally (1mg PCIII per kg mouse body weight) daily for 7 days following
6-OHDA intrastriatal stereotaxic injection (Figure 2A). Consistent with the previous report [16 19],
6-OHDA intoxication produced a phospho-serine 129 -synuclein (pS129- -Syn) positive Lewy like
inclusion in the TH-labeled dopaminergic neurons (Figure 2B). The PCIII treatment substantially
reduced pS129 -synuclein intensities (Figure 2B), which indicate prevention or degradation of
8]. In order to reproduce this observation in vivo, we used
,
α
,
α
α
α
6-OHDA-induced Lewy like inclusion formation.
The dopaminergic neuronal viability was assessed using immunohistochemistry with anti-tyrosine
hydroxylase (TH) specific antibodies. The TH-stained dopamine neurons in the substantia nigra
pars compacta were counted via unbiased stereological counting. The PBS-injected side of the
ventral midbrains showed approximately 4000 dopaminergic cells after i.p. administrations of either
DMSO or PCIII (Figure 2C,D). This result indicates that the PCIII treatment did not have overt
dopaminergic toxicity. On the other hand, PCIII treatment in 6-OHDA PD mouse models largely
alleviated the 6-OHDA-induced dopamine cell loss (Figure 2C,D). The loss of striatal dopaminergic
nerve terminals because of 6-OHDA intoxication in vivo was also substantially protected by PCIII
treatment, as demonstrated by TH optical density measurements in the striatum (Figure 2E,F).
We further evaluated the potential neuroinflammation using GFAP immunofluorescence, which labels
astrogliosis. The 6-OHDA injection increased GFAP immunofluorescence signal in the ventral midbrain
approximately three-fold higher than did the PBS injection in controls (Figure 2G,H). The PCIII
treatment reduced the GFAP signal (that was increased by 6-OHDA) by about two-fold compared
to that of the PBS injection control (Figure 2G,H). Taken together, these results demonstrated that
synthetic PCIII has neuroprotective functions in the 6-OHDA PD mouse model. The PCIII treatment
also prevented Lewy body-like structures of pS129-α-synuclein in dopamine neurons of the PD mouse
model. This result reflects the therapeutic potential of PCIII in PD treatment.

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Figure 2. PCIII prevents 6-OHDA-induced
α
-synuclein aggregation and dopamine cell death in vivo
.
(A) The schematic experimental schedule of PCIII therapeutic evaluation in 6-OHDA (8 µg) induced
PD mouse models. IHC, immunohistochemistry. To model the dopaminergic neuronal death in mice,
6-OHDA was stereotaxically injected into the striatum (coordinates from bregma, L: -2.0, AP: 0.5, DV:
-3.0 mm) of 2-month-old mouse brains. (
and serine 129-phosphorylated -synuclein (pS129
sections from 6-OHDA-induced PD mice treated with PCIII (i.p. 1mg/kg/day) or DMSO for seven
days. ( ) Representative tyrosine hydroxylase (TH) immunohistochemical staining of the substantia
nigra of the 6-OHDA PD mice treated with PCIII (i.p. 1 mg/kg/day, 7 days) or DMSO. ( ) Stereological
assessment of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra pars
compacta of the injection side of the indicated mouse groups (n = 4 mice per group). ( ) Representative
B
) Representative immunofluorescence images of TH (green)
α
α
-Syn, red) expression in the ventral midbrain
C
D
E
tyrosine hydroxylase (TH) immunohistochemical staining of the striatum (STR) of 6-OHDA PD mice
treated with PCIII (i.p. 1 mg/kg/day, 7 days) or DMSO. (F) Quantification of relative TH stained optical
dopaminergic nerve terminal fiber densities in the striatum of the indicated experimental groups (n = 4
mice per group). (
indicated mouse groups. (
nigra sections from the indicated experimental groups (n = 3 mice per group). The quantified data are
expressed as means SEMs. Statistical significance was determined by the ANOVA test with Tukey
G) Representative immunofluorescence images of GFAP in the substantia nigra of the
H
) Quantification of relative GFAP fluorescence intensities in the substantia
±
post-hoc analysis, ** p < 0.01, and *** p < 0.001. NS, nonsignificant.
2.3. Safety Profiling of Synthetic PCIII Administration In Vivo
Dopamine cell loss and Lewy body pathologies progress over several years in humans with
PD [1,20]. Therapeutic lead compounds would have to be administered for long periods of time in
order to adequately treat PD. Therefore, it is important to ascertain the safety of PCIII treatments
in vivo. As compared to the therapeutic dose of PCIII used in Figure 3, we administered 10 times
higher dose of PCIII (i.p. 10 mg/kg/day) for seven days to determine whether any toxicity is induced by
PCIII treatment in two-month-old C57/BL6 mice. We first measured the body weights of mice treated
with either DMSO vehicle or PCIII for seven days. There were no statistical differences in their body
weights (Figure 3A). There were also no differences between the spontaneous open field exploration

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in the two mouse groups (Figure 3B,C). Both groups showed similar total distances of exploration in
the open field arena during the 15-min periods (Figure 3B). The tendency to stay in the border of the
arena, as an indirect indicator of an anxiety phenotype, was not different between the DMSO and PCIII
treated mouse groups (Figure 3C). The groups also showed similar percentages of exploration distance
and time in each zone of the “center + periphery” and “border” (Figure 3C).
Figure 3. Safety profiling of PCIII in vivo. (
(i.p., 10 mg/kg/day) or DMSO vehicle for 7 days. (
A
) The body weights of mice treated with high dose PCIII
) The spontaneous exploration of mice treated with
B
high dose PCIII or DMSO traced by the open field test (left panel). The blue box is a center area, and the
red box is a periphery area. The area inside of the red box is “Center+Periphery” zone, and the area
outside of the red box is “Border” zone. The total distance traveled by mice treated with DMSO or
high dose PCIII during the 15-min session (n = 4 per group, right panel). (
assessed by comparing the time and distance spent between the center and periphery of the open
field arena (n = 4 mice per group). ( ) Motor coordination of the mice treated with high dose PCIII or
DMSO determined by rotarod test (n = 4 mice per group). ( ) Assessment of potential bradykinesia
determined by the pole test (n = 4 mice per group). The quantified data are expressed as means SEMs.
C) Relative anxiety was
D
E
±
Statistical significance was determined by unpaired two tailed student t-test test, NS, nonsignificant.
We next monitored the motor-related behaviors in mice that were treated with high dose PCIII.
Their motor coordination was assessed using an accelerated rotarod assay. After brief training on the
rotating rod, mice with DMSO or PCIII treatment were placed on the accelerating rotarod. We recorded
the latency time when the mice fell from the rotarod, and its speed at that time. Both groups failed to
show any statistical difference in motor coordination (Figure 3D). The pole test is commonly used to
assess bradykinesia. Both DMSO and PCIII-treated mice displayed similar performance in climbing
down the vertical pole (Figure 3E). These behavior experiments indicated the safety of PCIII, with no
obvious toxicity on the mice’s motor function.

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The potential tissue toxicities of high dose PCIII administration (i.p. 10 mg/kg/day, 7 days) were also
evaluated in six organs, including the heart, lung, liver, kidney, and spleen. Tissue distribution was first
determined using a developed bioanalytical method with ultra high-pressure liquid chromatography
(UHPLC) for deproteinated tissue homogenates. PCIII levels in each tissue are shown in Figure 4A.
PCIII in all six tissues was readily detectable. Importantly, the therapeutic target tissue, brain,
showed 230
the PCIII levels were found to be 231
162 48 ng/g tissue, 109 26 ng/g tissue for heart, lung, liver, kidney, and spleen, respectively
±
36 ng/g brain, suggesting that the PCIII is blood-brain barrier permeable. Also,
±
40 ng/g tissue, 121
±
12 ng/g tissue, 105 ± 3 ng/g tissue
,
±
±
(Figure 4A). However, the plasma concentration was observed to be under the lower limit of quantitation
(LOQ). With this distribution profile of PCIII in various mouse tissues, histological assessments was
performed to evaluate tissue toxicities using hematoxylin and eosin (H&E) staining. H&E staining
of these tissue sections from both mice groups revealed that high dose PCIII treatment did not
cause any structural alterations in vivo (Figure 4B). The potential PCIII toxicity in brain tissues was
determined using GFAP immunohistochemistry to monitor for any ongoing neuroinflammation. GFAP
immunostaining was conducted in several brain subregions of both mouse groups, including the
cortex, hippocampus, striatum, and ventral midbrain (Figure 4C,D). There was no difference in the
GFAP immunohistochemistry in these selected brain subregions of mice that were administered either
DMSO or PCIII (Figure 4C,D). These findings indicate that PCIII had no brain toxicity.
Figure 4. Tissue distribution and potential toxicity of PCIII in vivo. (
in each indicated tissue (ng/g tissue weight) determined by UHPLC (n = 3 mice for spleen and 4
mice for other tissues) ( ) Tissue toxicity profiling of PCIII with H&E staining of the indicated tissue
sections from mice treated with high dose PCIII (i.p., 10 mg/kg/day) or DMSO. ( ) Neuropathological
assessment of potential neuroinflammation with GFAP immunohistochemistry for the indicated brain
sections from mice treated with high dose PCIII (i.p., 10 mg/kg/day) or DMSO. Scale bar = 400
) Quantification of % area fraction occupied by GFAP positive signals in the IHC images of each
brain sections of mice treated with high dose PCIII or DMSO as control (n = 4 mice per group). The
quantified data are expressed as means SEMs. Statistical significance was determined by unpaired
two tailed student t-test test, NS, nonsignificant.
A) Quantification of PCIII levels
B
C
µm
(
D
±

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3. Discussion
Here we report the successful organic synthesis of PCIII racemate. PCIII synthesis was planned
using the basic esterification route of trans diol from seselin . Because the preparation routes to
were already established, we anticipated that large scale synthesis of
was possible. Among several known procedures, such as microwave-assisted cyclization,
], metal catalyzed cycloisomerization [10], and basic cyclization [11], the thermal
cyclization protocol was chosen for our scale-up procedure. This protocol was chosen because of its
easily handled purification protocol and high chemical yield. With synthetic seselin in a decagram
scale in hand, trans diol was produced after m-CPBA mediated epoxidation and the aqueous
epoxide opening sequence. We then attempted to perform pivotal selective esterification of the
trans diol . Because two alcohols were secondary alcohols, it was difficult to predict which alcohol
5
4
seselin
seselin
4 from the umbelliferone 2
4
thermal cyclization [
9
4
5
5
moiety would react dominantly. In a structural view, the benzylic alcohol is similar to an aromatic
ring. Inside the structure, another alcohol is neighbored by a sterically demanding gem-dimethyl
group. When tigloylation was carried out under a DMAP-assisted condition, two constitutional
isomers
the spectral analysis and document search showed that the undesired isomer
Regarding less reactivity of benzylic alcohol moiety, acetylation/tigloylation of the trans diol
7
and
8
were obtained and purified after careful silica gel chromatography. Unfortunately,
was a major product.
sequence
7
5
was planned. The standard acetylation (Ac2O, iPr2NEt, DMAP) condition, however, afforded an
inseparable mixture of corresponding acetyl isomers along with an unreacted starting material.
Because the desired esterification route (acetylation/tigloylation) was unsuccessful, the early procedure
(tigloylation/acetylation) route was chosen for large scale synthesis of the desired tigloyl ester
However, this procedure had low isolation yield. Finally, the simple acetylation of the desired tigloyl
ester produced PCIII as a racemic mixture. This synthetic PCIII was applied for further biological
8.
8
1
evaluations, giving similar anti-aggregate activity compared to purified PCIII. It is still a mystery
how the synthetic PCIII racemate produced similar therapeutic potential to that of purified PCIII.
It would be beneficial to design and validate the organic PCIII synthesis of identical optical stereoisomer
structures with purified PCIII. This future study would provide further understanding of the structure
and function relationship of PCIII’s stereoisomers and their modes of binding to amyloid protein
aggregate. In addition, our successful organic synthesis of PCIII provided reactive intermediates.
These intermediate compounds could be applied to compare the structure-activity relationship. They
can also be used to synthesize the structural analogs of PCIII to improve its physicochemical properties
or therapeutic function.
PCIII was first evaluated for its therapeutic potential in PD mouse models. With only 1 mg/kg
administration of PCIII, 6-OHDA induced dopaminergic cell loss and
diminished substantially. PCIII has been reported to bind protein aggregates, such as
aggregate to disentangle them. In doing so, PCIII facilitates proteasomal degradation of protein
aggregates [ ]. Consistent with previous findings, we showed that in vivo PCIII inhibits pS129- -Syn-
α
-synuclein aggregation were
α
-synuclein
8
α
positive Lewy like inclusions in dopamine neurons of PD mouse model. It should be noted that
dopaminergic neuroprotection by PCIII administration was not complete compared to the marked
inhibition of
PCIII might be due to oxidative stress and other signaling alterations caused by 6-OHDA stress.
However, this in vivo result is consistent with partial cytoprotection by PCIII in -synuclein-expressing
α-synuclein aggregation. This partial dopaminergic rescue by anti-aggregate compound
α
SH-SY5Y cells challenged with 6-OHDA (Supplementary Figure S1B). This finding suggests the need
to apply multiple medications targeting different pathogenic alterations to better control PD. Since
PCIII targets
α-synucleinopathies on various brain subregions. Therefore, future studies are needed to investigate
the role of PCIII on other -synuclein transgenic mice or preformed fibril -synuclein injection mouse
α-synuclein aggregates, it is possible to use PCIII in disease-modifying therapies of
α
α
models. An important finding of our study is the in vivo safety validation of PCIII with high dose
treatment. Seven days of high dose PCIII administration had no obvious toxic effect on various organs.
In addition, PCIII treatment did not cause any inflammatory alterations in diverse brain subregions.

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At the behavior level, PCIII had no adverse effect on the motor coordination or muscle fine control.
Compared to the 1 mg/kg therapeutic dose of PCIII, a 10-times higher dose of PCIII was not found to
have any obvious toxicity in vivo. The high therapeutic index of PCIII is advantageous in the treatment
of neurodegenerative disorders, which often require repeated medications during disease progression.
It is still necessary to test higher doses of PCIII which may give overt toxicity in vivo in order to outline
PCIII’s safety index. It would be also critical to evaluate higher dose PCIII treatment in PD mouse
models to see if the disease models tolerate the drug with expected therapeutic effect.
Although extensive pharmacokinetic studies of PCIII in the preclinical animals were not performed
in this study, we found that PCIII distributed to the brain, the therapeutic target organ, with a
concentration of 230
±
36 ng/g mouse brain (Figure 4A). The extent of tissue distribution of PCIII to brain
was higher than those to the liver and lung of mice with 7 days of PCIII treatment. Considering that the
blood-brain barrier permeability is an important factor for any compound targeting neurodegenerative
diseases, this finding of PCIII distribution suggests that PCIII might be a promising new drug candidate
for PD treatment. However, the plasma concentration of PCIII was found to be under lower detection
limit of quantitation (i.e., 20 ng/mL). This low plasma concentration of PCIII despite demonstrable
distribution of PCIII in other organs remained to be investigated in the future. Moreover, the extensive
pharmacokinetic studies including metabolic stability of PCIII may provide instructive translational
information prior to clinical evaluation of this drug.
In conclusion, synthetic PCIII racemate holds a great translational value in the treatment of
PD pathogenesis including dopaminergic neuron loss and α-synuclein aggregation. It is, however,
required to investigate pharmacokinetic properties of PCIII and its target protein binding mode before
clinical application.
4. Materials and Methods
4.1. Chemicals and Antibodies
The PCIII was provided from the National Development Institute of Korean Medicine (NIKOM,
Gyeongsan, Korea), as previously described [
from Sigma. The following primary antibodies were used: mouse antibody to FLAG (M2 clone,
1:5,000, Sigma), mouse antibody to pS129- -synuclein (#825702, 1:3000, Biolegend, San Diego, CA,
8]. 6-OHDA, goat serum, and doxycycline were purchased
α
USA), rabbit antibody to tyrosine hydroxylase (NB300-109, 1:2000, Novus Biologicals, Centennial, CO,
USA), and rabbit antibody to GFAP (#ab7260, 1:1,000, Abcam, Cambridge, MA, USA). The following
secondary antibodies were also used: horse radish peroxidase (HRP)-conjugated sheep antibody
to mouse IgG (cat# RPN4301, 1:5000, GE Healthcare, Pittsburgh, PA, USA), biotin-conjugated goat
antibody to rabbit IgG (cat# BA-1000, 1:1000, Vector Laboratories, Burlingame, CA, USA), Alexa Fluor
488-conjugated goat antibody to rabbit IgG (cat# A11034, 1:1,000; Invitrogen, Carlsbad, CA, USA),
Alexa Fluor 568-conjugated donkey antibody to mouse IgG (cat# A11031, 1:1,000; Invitrogen, Carlsbad,
CA, USA), and HRP-conjugated mouse antibody to
St. Louis, MO, USA).
β-actin (cat# A3854, 1:10000, Sigma-Aldrich,
4.2. Organic Synthesis of PCIII
The general procedure was performed as follows. For better understanding by readers, we included
step-by-step procedure images which were divided and extracted from Figure 1A (full organic synthesis
procedure of PCIII) in this method section. Unless noted otherwise, all of the starting materials and
reagents were obtained from commercial suppliers and were used without further purification.
Tetrahydrofuran and Et2O were distilled from sodium benzophenone ketyl. Dichloromethane,
triethylamine, acetonitrile, and pyridine were freshly distilled from calcium hydride. All of the
solvents used for routine product isolation and chromatography were reagent grade and glass distilled.
The reaction flasks were dried at 100 ◦C. The air and moisture sensitive reactions were performed
under argon atmosphere. The chemical shifts are expressed in parts per million (ppm, δ) downfield

Int. J. Mol. Sci. 2019, 20, 5481
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from tetramethylsilane and are referenced to the deuterated solvent (CHCl3). The 1H-NMR data were
reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet and/or multiple resonance), number of protons, and the coupling constant in hertz (Hz).
To a solution of umbelliferone
2 (8.6 g, 53 mmol) in p-xylene (100 mL), 1,1-diethoxy-3-methyl-
2-butene (10 g, 64 mmol) and 3-picoline (1.3 mL, 13 mmol) were added and refluxed. After 24 h,
3
the reaction flask was cooled, diluted with CH2Cl2, and filtered. The organic residue was evaporated
in vacuo and the residue was purified using column chromatography on silica gel (EtOAc: n-hexane =
1:5) to present seselin 4 (9.6 g, 80%) as an amorphous solid.
1
4
: H-NMR (CDCl3, 300 MHz)
δ
7.60 (1H, d, J = 9.6 HZ), 7.20 (1H, d, J = 8.7 Hz), 6.88 (1H, dd, J =
0.6, 8.4 Hz), 6.72 (1H, dd, J = 0.6, 8.4 Hz), 6.23 (1H, d, J = 9.6 Hz), 5.73 (1H, d, J = 9.9 Hz), 1.48 (6H, s);
13C-NMR (CDCl3, 75 MHz)
161.0, 155.3, 150.1, 143.9, 130.7, 127.7, 114.9, 113.5, 112.5 (2C), 109.2, 77.6,
28.1 (2C) HRMS (EI): Calcd. for C14H12O3 ([M]+): 228.0786, found: 228.0783.
δ
To a solution of seselin
4 (89 mg, 0.39 mmol) in CH2Cl2 (3 mL), mCPBA (77%, 96 mg, 0.43 mmol)
and NaHCO3 (164 mg, 1.95 mmol) were added at 0 ◦C. After 5 h of stirring at room temperature, H2O
was added and extracted with CH2Cl2 twice. The organic layers were dried over MgSO4, filtered,
and evaporated in vacuo. The residue was used in the next reaction without further purification.
The residue was dissolved in acetone (8 mL). Next, H2SO4 (1M, 4 mL, 4 mmol) was added to the
reaction flask and stirred at room temperature for 1 h. After aq. NaHCO3 was added to the reaction
mixture, it was transferred to a separatory funnel and extracted with EtOAc twice. The organic
layers were dried over MgSO4, filtered, and evaporated in vacuo. The residue was purified using
column chromatography on silica gel (EtOAc: n-hexane = 1:1) to afford diol
5 (57 mg, 55%) as an
amorphous solid.
1
5
: H-NMR (CDCl3, 300 MHz)
δ
7.66 (1H, d, J = 12.3 HZ), 7.32 (1H, d, J = 8.7 Hz), 6.79 (1H, d, J =
8.7 Hz), 6.26 (1H, d, J = 9.6 Hz), 5.04 (1H, d, J = 6.6 Hz), 3.88 (1H, d, J = 6.6 Hz), 3.66 (2H, br), 1.54 (3H,
s), 1.32 (3H, s); 13C-NMR (CDCl3, 75 MHz)
161.1, 156.2, 154.2, 144.4, 128.5, 114.8, 112.4, 112.1, 111.7,
79.4, 74.8, 66.5, 25.5, 20.0. HRMS (EI): Calcd. for C14H14O5 ([M]+): 262.0841, found: 262.0823.
δ
To a solution of diol
5 (52 mg, 0.2 mmol) in CH2Cl2 (3 mL), iPr2NEt (0.070 mL, 0.40 mmol), tigloyl
chloride
6
(0.026 mL, 0.24 mmol) and DMAP (2.5 mg, 0.02 mmol) were added at 0 ◦C. The reaction

Int. J. Mol. Sci. 2019, 20, 5481
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mixture was warmed to room temperature and stirred for six hours. After adding aq. NH4Cl to the
reaction mixture, it was transferred to a separatory funnel and extracted with CH2Cl2 twice. The
organic layers were dried over MgSO4, filtered, and evaporated in vacuo. The residue was purified by
column chromatography on silica gel (EtOAc: n-hexane = 1:2) to afford 7 (27 mg, 39%), and 8 (3mg,
4.4%) as amorphous solids.
1
7
: H-NMR (CDCl3, 300 MHz)
δ
7.62 (1H, d, J = 9.6 HZ), 7.30 (1H, d, J = 8.1 Hz), 6.78 (1H, d, J =
8.4 Hz), 6.76 (1H, ddd, J = 12.3, 5.4, 1.8 Hz), 6.21 (1H, d, J = 9.3 Hz), 5.20 (1H, d, J = 3.3 Hz), 5.04 (1H, d,
J = 3.3 Hz), 1.76 (3H, s), 1.72 (3H, dd, J = 7.2, 1.0 Hz), 1.47 (3H, s), 1.38 (3H, s); 13C-NMR (CDCl3, 75
MHz)
δ 166.8, 160.8, 155.9, 154.5, 144.0, 139.5, 128.5, 128.0, 114.7, 112.5, 112.4, 110.4, 77.3, 74.0, 62.8, 23.9,
23.8, 14.4, 12.0. HRMS (EI): Calcd. for C19H20O6 ([M]+): 344.1260, found: 344.1266.
1
8
: H-NMR (CDCl3, 500 MHz)
δ
7.58 (1H, d, J = 9.5 HZ), 7.33 (1H, d, J = 8.0 Hz), 6.84 (1H, d, J =
7.0 Hz), 6.79 (1H, d, J = 8.5 Hz), 6.21 (1H, d, J = 9.5 Hz), 6.08 (1H, s), 3.93 (1H, s), 3.36 (1H, br), 1.82 (3H,
s), 1.75 (3H, dd, J = 7.0 Hz), 1.46 (3H, s), 1.37 (3H, s); 13C-NMR (CDCl3, 75 MHz)
δ
168.9, 160.1 157.0,
154.3, 143.4, 139.0, 129.1, 128.0, 114.6, 113.0, 112.5, 107.4, 79.1, 73.2, 68.5, 24.7, 21.3, 14.5, 12.1. HRMS
(EI): Calcd. for C19H20O6 ([M]+): 344.1260, found: 344.1256.
To a solution of 7 (4 mg, 0.012 mmol) in CH2Cl2 (2 mL), iPr2NEt (6 drops, excess), acetic anhydride
(4 drops, excess), and DMAP (1 mg, 0.08 mmol) were added at 0 ◦C. The reaction mixture was warmed
to room temperature and stirred for six hours. After adding aq. NH4Cl to the reaction mixture, it was
transferred to a separatory funnel and extracted with CH2Cl2 twice. The organic layers were dried
over MgSO4, filtered, and evaporated in vacuo. The residue was purified by column chromatography
on silica gel (EtOAc: n-hexane = 1:2) to afford PCIII 1 (4 mg, 89%) as an amorphous solid.
1
PCIII
1
: H-NMR (CDCl3, 500 MHz)
δ
7.57 (1H, d, J = 9.5 HZ), 7.35 (1H, d, J = 9.0 Hz), 6.81 (1H, d,
J = 8.5 Hz), 6.78 (1H, dq, J = 7.5, 1.0 Hz), 6.20 (1H, d, J = 8.5 Hz), 6.20 (1H, d, J = 3.0 Hz), 5.30 (1H, d, J
= 3.5 Hz), 2.06(3H, s), 1.82 (3H, t, J = 1.0 Hz)), 1.73 (3H, dd, J = 7.0, 1.0 Hz), 1.44 (3H, s), 1.36 (3H, s);
13C-NMR (CDCl3, 125 MHz)
δ 169.3, 166.3, 159.8, 156.5, 154.3, 143.1, 138.0, 129.1, 128.1, 114.4, 113.3,
112.4, 106.6, 77.0, 71.3, 63.3, 23.8, 23.7, 20.7, 14.4, 12.1. HRMS (EI): Calcd. for C21H22O7 ([M]+): 386.1366,
found: 386.1348.
4.3. Cell Culture and Transfection
We maintained human neuroblastoma SH-SY5Y cells (ATCC, Manassas, VA) in DMEM containing
10% FBS (v/v) and antibiotics (penicillin-streptomycin 100 U/mL, ThermoFisher Scientific). The
cell culture incubator was set with a humidified atmosphere consisting of 5% CO2/95% air at
37 ◦C. To transiently transfect the indicated plasmids (pCMV-tTA, TetP-
HA- -synuclein [
β
23, pTet-dual2, and
α
8]) to SH-SY5Y cells, the X-tremeGENE HP transfection reagents (Roche) were
used following the manufacturer’s protocol.
4.4. Fluorescence Imaging
The SH-SY5Y were plated onto poly-D-lysine-coated coverslips at a density of 10,000 cells/cm2.
Following the experimental procedures (Figure 1B), we fixed the cells with 4% paraformaldehyde
in PBS and blocked in a solution containing 5% normal goat serum (Sigma-Aldrich, St. Louis, MO,
USA) and 0.1% Triton X-100 (Sigma) for 1 h at room temperature. The fixed cells were then i◦ncubated
overnight with their corresponding primary antibodies against FLAG-tagged
β23 at 4 C. After

Int. J. Mol. Sci. 2019, 20, 5481
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three brief washes with PBS containing 0.1% Triton X-100, we incubated the samples for one hour
with fluorescence-conjugated secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA) at room
temperature. Coverslips were then mounted with 4’,6-diamidino-2-phenylindole (DAPI) for nuclear
counter staining. The fluorescent images acquisition was done by using a fluorescence microscope
(Axiovert, 200 M; Carl Zeiss, Germany).
4.5. Western Blotting
For the preparation of total protein lysates, we added lysis buffer (1% Nonidet P40 in phosphate-
buffered saline (PBS), pH 7.4) that was supplemented with protease/phosphatase inhibitors to the
SH-SY5Y cells after a brief wash with ice cold PBS. After three freeze and thaw cycles in dry ice and
iced water for complete lysis of cells, respectively, the samples were centrifuged at 14,000
The supernatants were mixed with the same volume of 2X Laemmli buffer (Bio-Rad, Hercules, CA,
USA) supplemented with -mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), and boiled for
×
g for 30 min.
β
five minutes. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes
for immunoblotting. The blotted nitrocellulose membranes were stained with Ponceau (Sigma) to
verify the uniform protein transfer. The nitrocellulose membrane was incubated with the designated
antibodies, and immunoreactive bands were visualized via chemiluminescence (Pierce). Next, we
performed densitometric analyses of the bands using ImageJ (NIH, http://rsb.info.nih.gov/ij/).
4.6. Cell Viability Assay
We plated SH-SY5Y cells on 6-well plates at a density of 0.5
×
106 cells per well. Following transient
transfection with the indicated constructs and subsequent treatment (Figure 1B, and Supplementary
Figure S1), the cells were harvested as single cell suspensions by trypsinization. After two PBS washes,
the cells were resuspended in serum-free DMEM. The resuspended cells were mixed with an equal
volume of 0.4% trypan blue (w/v) and incubated for 2 min at room temperature. Live and dead cells
were counted using a Countess II Automated Cell Counter (Life Technologies, Bothell, WA, USA) for
cell viability measurement.
4.7. Animal Experiments
All of the animal experiments were approved by the Ethical Committee of Sungkyunkwan
University (Approval #: SKKUIACUC2019-06-15-1; Approval date: 2019-07-02) and were conducted
in accordance with all applicable international guidelines. Male C57BL/6N mice (2 months old) was
obtained from Orient Bio (Suwon, Korea). The animals were housed in air-controlled rooms with a 12-h
dark/light cycle. Mice and rats were provided ad libitum access to diet and water. All efforts were made
to minimize the animals’ suffering and the number of animals used in experiments. Synthetic PCIII
racemate was administered to mice intraperitoneally (i.p.). The i.p. PCIII administration (1 mg/kg/day
for 6-OHDA PD mouse models; 10 mg/kg/day for tissue distribution and toxicity experiments) began
on day 0 and was continued for 7 days. Intrastriatal injection of 6-OHDA was performed on day 0.
The mouse brains were prepared for analysis as described below. To determine the tissue distribution
and toxicity of PCIII in vivo, the brains and other biological tissue samples were collected at 30 min
after the last administration of PCIII.
4.8. UHPLC Bioanalytical Method of PCIII
For the determination of levels of tissue concentration for PCIII, the mouse tissues including
brain were homogenized by adding two-fold volume of PBS, and then the tissue homogenates
were deproteini◦zed by adding two-fold volume of acetonitrile. After centrifugation at 15,000 rpm
for 15 min at 4 C, the supernatant was taken for the next analysis. A UHPLC-DAD method for
PCIII was established using an UHPLC system comprising of the Agilent 1290 Infinity II UHPLC
system (Agilent Technologies, Santa Clara, CA, USA), equipped with an auto-sampler (G7167B),
a flexible pump (G7104A), a Multicolumn Thermostat (MCT) (G7116B), and a DAD detector (G7117A).

Int. J. Mol. Sci. 2019, 20, 5481
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The separation between PCIII and endogenous substances derived from plasma was accomplished
using a CapcellpakTM C18 5
m column (UG120; 250 1.5 mm, Shiseido Fine Chemical Co., Tokyo,
µ
×
Japan). A gradient condition was applied with a mixture of 0.1% phosphoric acid and acetonitrile
at a flow rate 0.2 mL/min. The percentage of mobile phase (0.1% phosphoric acid and acetonitrile)
was as follows; from initial to 1 min, 50:50; from 1 to 3 min, 15:85; from 3 to 7 min, 15:85; from 7 to
9 min, 50:50; from 9 to 16 min, 50:50. The DAD detector was set to 321 nm and injection volume was
10 µL. Calibration curve was set up in a range covering from 20 to 1000 ng/mL for PCIII. The linearity,
precision, and accuracy for PCIII were observed within the acceptable ranges. The tissue concentration
level was expressed as a unit of PCIII amount (ng)/g tissue, as described previously [21].
4.9. Intrastriatal Injection of 6-OHDA
Intrastriatal injeciton of 6-OHDA was performed as previously described [22] with some
modifications. For stereotaxic injection of 6-hydroxy dopamine (6-OHDA, 8
µg), 2-month-old
C57/BL6N mice treated either with PCIII for 7 days or DMSO as a control were anesthetized with
alfaxalone (100 mg/kg). An injection cannula (26.5 gauge) was applied stereotaxically into the striatum
(anteroposterior, 0.5 mm from bregma; mediolateral, 2.0 mm; dorsoventral, 3.0 mm) and unilaterally
applied into the right hemisphere. The infusion rate was 0.2
µl/min with total injection volume of
2
µ
l of 6-OHDA (4 g/ l in sterile PBS) into each mouse. After the 6-OHDA injection was completed,
µ µ
the cannula was maintained in the striatum for an additional 5 min to facilitate complete absorption of
the 6-OHDA to brain tissues. The cannula was then slowly removed from the mouse brain. The skin over
the skull was closed by suturing. We closely monitored wound healing and recovery of mice following
the surgery. For the stereological analysis, the animals were perfused at 7 days after intrastriatal
6-OHDA injection and fixed intracardially with ice-cold PBS followed by 4% paraformaldehyde. The
tissue was post-fixed with 4% paraformaldehyde overnight. The fixed mouse brains were cryoprotected
in 30% sucrose (w/v) in PBS and subsequently processed for immunohistochemistry.
4.10. TH Stereological Cell Counting and Immunohistochemistry
TH setereological assessment was carried out as previously described [22]. Briefly, coronal sections
(thickness of 40 um) of the fixed and cryoprotected brains were cut, including the substantia nigra.
Every fourth section was used for analysis. To analyze the tyrosine hydroxylase (TH) expression,
the sections were incubated with a 1:1000 dilution of rabbit polyclonal anti-TH (Novus) antibody
followed by sequential incubations with biotinylated goat anti-rabbit IgG and streptavidin-conjugated
horseradish peroxidase (HRP) using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA)
according to the manufacturer’s instructions. To visualize the TH-positive cells, 3,3-diaminobenzidine
(DAB, cat# D4293, Sigma-Aldrich, St. Louis, MO, USA) was used as an HRP substrate. The total
number of TH-positive neurons in the substantia nigra pars compacta was determined using the
Optical Fractionator probe in Stereo Investigator software (MicroBrightfield, Williston, VT, USA).
All of the stereological counting was performed in a blinded manner to the mouse treatments.
For immunofluorescence labeling, coronal ventral midbrain sections were blocked in PBS containing 4%
normal goat serum and 0.2% Triton-X for 1 h at room temperature. The brain sections were incubated
with the indicated antibodies overnight at 4 ◦C, followed by a brief washing and subsequent incubation
in PBS containing fluorescent dye labeled secondary antibodies. The fluorescent images were obtained
using a fluorescence microscope (Axiovert, 200 M; Carl Zeiss, Germany) for image acquisition.
4.11. Behavior Tests
A rectangular wood box (40 cm
5 cm) was used as an open arena for the open field behavior test. The field was subdivided
into border, peripheral, and central sectors automatically by a video tracking system (Smart v3.0
software). The central region included 4 central squares (2 2), the peripheral region included 12
squares surrounding the center region, and the border regions made up the remaining squares. The
×
40 cm
×
40 cm) divided into 64 (8
×
8) identical regions (5
cm
×
×

Int. J. Mol. Sci. 2019, 20, 5481
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mouse was placed at the center of an open field and allowed to explore for 15 min under dim light.
We cleaned the open field arena with diluted 70% ethanol between each trial. A video tracking system
(Smart v3.0 software) was used to record the distance traveled as a measure of locomotor activity.
The time spent in and entries into the center were measured as an anxiolytic indicator. The mice were
trained 2 days before the rotarod test. On day 3, mice were placed on an accelerating rotarod cylinder,
and the latency time at which they fell off the machine was measured. The speed of the rotarod was
slowly increased from 4 to 40 rpm within 5 min. A trial ended if the animal fell from the rotarod.
The test data are presented as means of latency time (3 trials). For the pole test, the animals were
allowed to acclimate to the behavioral procedure cage for at least 30 min. The pole is made up of 58 cm
of metal rod with 10 mm diameter with bandage gauze wrapped around the pole. Briefly, the pole was
placed inside the acclimatized cage, and the mice were placed on the top portion of the pole facing
head-down. The total time that it took the animals to reach the base of the pole was recorded. The mice
were evaluated in three sessions, and the average time was recorded.
4.12. Tissue Pathological Staining
4% PFA fixed and 30% sucrose embedded tissue sections were sliced at 40 um thickness for
pathological assessment. The tissue sections mounted onto glass slides (Fisher Scientific) were processed
for hematoxylin and eosin (H&E) staining. After dehydration step, the sections were stained with
hematoxylin (Merck, Darmstadt, Germany) for 3 min and eosin (Merck, Darmstadt, Germany) for
1 min, respectively. The sections were destained by rinsing with gradually increasing the concentrations
of ethanol/water solutions and then mounted with DPX mounting medium (Sigma-Aldrich) after
the fixation in xylene. For the neuropathological assessments, GFAP immunohistochemistry was
performed by the procedures described in the method Section 4.10. Bright field images were obtained
using a microscope (Axiovert, 200 M; Carl Zeiss, Germany).
4.13. Statistics
The quantitative data are presented as means
±
SEMs. Power analysis was done by using G*Power
3.1 software to estimate the approximate sample sizes for tyrosine hydroxylase stereological counting.
On the basis of the mean difference from our preliminary experiments and related previous report [22],
a total sample size of four mice was calculated to potentially obtain significant differences (effect
size f = 22.42 for 45% mean difference;
α = 0.05). The statistical significance was assessed using an
analysis of variance (ANOVA) test with Tukey’s HSD post hoc analysis (comparisons of more than
three groups). Differences with a p value < 0.05 were considered statistically significant. We used
GraphPad Prism software to prepare all of the plots and statistical analyses.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/20/21/
5481/s1.
Author Contributions: Y.L., S-M.P., and H-J.M. conceived the hypothesis and supervised the project. S.H.
performed the cellular experiments to compare the biological functions of synthetic and organic PCIII. S.H. also
performed in vivo 6-OHDA injection studies. H.K. (Heejeong Kim) performed toxicity studies for high doses of
PCIII treatment. B.S. and S-M.P. synthesized the organic PCIII racemate. J.-H.Y., and H.-J.M. analyzed the tissue
distribution of PCIII by using UHPLC. S.H., H.K. (Heejeong Kim) and H.K. (Hyojung Kim) contributed to the
data analysis and figure preparation. Y.-S.L., and J.-Y.C. provided helpful discussion regarding experimental
design. Y.L., S.-M.P., and H.-J.M. wrote the manuscript. All of the authors contributed to the final review of the
manuscript and figures.
Funding: This research was supported by grants from the National Research Foundation of Korea
(NRF-2017M3C7A1043848) funded by the Korea Ministry of Science, ICT, & Future Planning (MSIP). This
research was also supported by the Basic Science Research Program through the National Research Foundation
of Korea (NRF), funded by the Ministry of Education, Science and Technology (NRF-2016R1C1B2006699,
NRF-2019R1F1A1058103).
Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Mol. Sci. 2019, 20, 5481
15 of 16
Abbreviations
PD
Ac2O
Parkinson’s disease
Acetic anhydride
m-CPBA meta-Chloroperoxybenzoic acid
6-OHDA 6-hydroxydopamine
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