H.I. Kim et al.
Pharmacological Research 165 (2021) 105423
BDNF acts as a trophic factor that regulates survival and phenotypes
anti-p-JNK (#9251S), rabbit-anti-mTOR (#2972S), rabbit-anti-p-
of the mesencephalic dopaminergic (DAergic) neurons [8,9]. Further-
more, it increases the number of tyrosine hydroxylase (TH)-positive
neurons and DA secretion [10] in the striatum and substantia nigra [8,
11]. In fact, significant striatal neuronal loss was detected in Bdnf
knockout mice [12], and morphological and functional changes in the
striatum with motor dysfunction were observed in Bdnf and TrkB mutant
mice [11,13]. Recent evidences also demonstrated that BDNF promotes
neuroprotection and neuroregeneration [14]. DAergic neurons in the
substantia nigra of patients with Parkinson’s disease (PD) show low
BDNF mRNA expression levels, which might pose a significant risk in PD
pathology [15]. Thus, BDNF regulatory signals are potential therapeutic
targets for neurodegenerative diseases, including PD.
mTOR (#2971S), rabbit-anti-GAPDH (#2118), mouse-anti-α-tubulin
(T6074), and mouse-anti-β-actin (A1978) (all from Cell Signaling
Technology, Danvers, MA, USA); mouse anti-horseradish peroxide
(HRP)-conjugated anti-rabbit, anti-mouse, and anti-rat IgG (all from
Thermo Fisher Scientific). Alexa Fluor®-conjugated secondary anti-
bodies were from Invitrogen (Waltham, MA, USA). Goat anti-rabbit
biotinylated secondary antibody was from Vector Laboratories (Burlin-
game, CA, USA).
HPB2 was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St.
Louis, MO, USA) to prepare a 50 mM stock solution, which was stored at
-20 ◦C. GSK4716, GSK5182, PD98059, SB203580, SP600125, and ANA-
12 were purchased from Sigma-Aldrich. All compounds were dissolved
in DMSO.
The estrogen-related receptors (ERRs) of the orphan nuclear receptor
family consist of three members, ERRα (NR3B1), ERRβ (NR3B2), and
ERRγ (NR3B3) [16]. ERRγ is expressed in the embryo and adult tissues,
such as the brain, skeletal muscle, heart, and liver, where it regulates
metabolic signals by acting as a transcription factor, growth factor, and
hormone [17–19]. In the developing mouse brain, ERRγ is expressed
from E10.5, and its level in the floor of the mesencephalon increases at
E11.5 during neuronal differentiation [19]. ERRγ is widely expressed in
the adult mice brain, including the olfactory bulb, cerebral cortex,
hippocampus, thalamus, hypothalamus, midbrain, striatum, amygdala,
and brain stem [20]. Despite the abundant expression of ERRγ in the
nervous system, its biological role in the nervous system is largely un-
known. Previously, we showed for the first time the relevance of ERRγ in
the regulation of DAergic neuronal phenotype—ERRγ is upregulated
during DAergic neuronal differentiation and is involved in the regula-
tion of DAergic neuronal marker TH and dopamine transporter (DAT)
and impacts neuronal morphology including neurite outgrowth [21].
Based on the evidence that both ERRγ and BDNF play crucial roles in
the regulation of the DAergic neuronal phenotype, the present study is
aimed to determine whether ERRγ is involved in the regulation of BDNF,
which was responsible for ERRγ-induced enhancement of the DAergic
phenotype. Toward this, we synthesized the ERRγ ligand derivative,
HPB2, based on ERRγ structure and computational docking simulation
study, and examined the effects of HPB2 on the regulation of BDNF and
DAergic neuronal phenotype via relevant signaling pathways in DAergic
cell lines and primary cultured ventral mesencephalic (VM) neurons. We
also verified the effect of HPB2 on BDNF levels and DAergic phenotype
in vivo. As BDNF is emerging as a new therapeutic target for various
neurological and degenerative brain diseases, our evaluation of the
relevance of ERRγ in the regulation of BDNF and subsequent DAergic
maturation might help develop novel therapeutic strategies for these
diseases.
2.2. Synthesis of HPB2
4-(Phenylethynyl)benzaldehyde: To a stirred solution of 4-bromo-
benzaldehyde (2.50 g, 13.5 mmol, 1.0 equiv.) and phenylacetylene
(2.97 ml, 27.0 mmol, 2.0 equiv.) in anhydrous tetrahydrofuran (THF)
(100 mL) was added bis(triphenylphosphine)palladium(II) dichloride
(Pd(PPh3)2Cl2) (473 mg, 0.675 mmol, 0.05 equiv.), CuI (257 mg, 1.35
mmol, 0.1 equiv.), and Et3N (3.77 mL, 27.0 mmol, 2.0 equiv.). The re-
action mixture was heated to reflux overnight. The reaction mixture was
cooled to room temperature and water (30 mL) was added and extracted
with EtOAc (twice, 100 mL). The combined organic layer was washed
with water (10 mL) and brine (10 mL), dried over MgSO4, and evapo-
rated under reduced pressure. The crude mixture was purified using
silica gel column chromatography (EtOAc: hexane = 1: 10) to form a
1
white solid (2.67 g, 13.0 mmol) with 96 % yield. H-NMR (500 MHz,
CDCl3) δ 10.00 (s, 1 H), 7.85 (d, J =8.6 Hz, 2 H), 7.66 (d, J =8.1 Hz, 2 H),
7.56-7.53 (m, 2 H), 7.38-7.36 (m, 3 H); 13C NMR (125 MHz, CDCl3) δ
191.5, 135.4, 132.2, 131.8, 129.6, 129.0, 128.5, 122.5, 93.5, 88.6;
HRMS (ESI+) found 207.0806 [calculated for C15H11O ([M]+):
207.0804].
(E)-4-hydroxy-Nʹ-(4-(phenylethynyl)benzylidene)benzohydrazide
(HPB2): 4-hydroxy benzohydrazide (1.14 g, 7.46 mmol, 1.0 equiv.) was
added to a stirred solution of benzaldehyde (2.0 g, 9.70 mmol, 1.3
equiv.) in n-BuOH (100 mL) and the mixture was heated to reflux for 10
h. The reaction mixture was cooled to room temperature and n-BuOH
was removed under reduced pressure. MeOH was added to remove re-
sidual n-BuOH. The crude mixture was adsorbed on silica gel and puri-
fied using silica gel column chromatography (dichloromethane: MeOH
= 10:1). EtOAc was added to the compound, and the solution was
sonicated and filtered to obtain HPB2 (2.16 g, 6.34 mmol) with 85 %
yield as an off-white solid. 1H-NMR (800 MHz, DMSO-d6) δ 11.77 (s, 1
H), 10.16 (s, 1 H), 8.46 (s, 1 H), 7.84 (d, J =8.5 Hz, 2 H), 7.76 (d, J = 5.8
Hz, 2 H), 7.61 (d, J =8.0 Hz, 2 H), 7.58-7.56 (m, 2 H), 7.44-7.42 (m, 3
H), 6.88 (d, J =8.6 Hz, 2 H); 13C-NMR (800 MHz, DMSO-d6) δ 162.8,
160.8, 145.7, 134.7, 131.8, 131.4, 129.7, 128.9, 128.7, 127.1, 123.7,
123.3, 122.1, 115.0, 90.9, 89.2; HRMS (ESI+) found 341.1281 [calcu-
lated for C22H17N2O2 ([M]+): 341.1285].
2. Materials and methods
2.1. Antibodies and reagents
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum
(FBS), and penicillin/streptomycin were purchased from Corning
(Corning, NY, USA). Trypsin and ethylenediaminetetraacetic acid were
purchased from Cytiva (Marlborough, MA, USA). DMEM/F12 medium,
N2 supplement, neurobasal medium and B27 supplement were pur-
chased from Thermo Fisher Scientific (Waltham, MA, USA). The
following antibodies were used: rabbit-anti-BDNF (sc-546), rabbit-anti-
p-CREB (sc-7989), rat-anti-DAT (sc-33258), mouse-anti-TH (sc-25269)
rabbit-anti-ERK (sc-154), mouse-anti-p-ERK (sc-7383), mouse-anti-Akt1
(sc-271149), and mouse-anti-vinculin (sc-73614) (all from Santa Cruz
Biotechnology, Dallas, TX, USA); rabbit-anti-TrkB (GTX54857), rabbit-
anti-p-TrkB (GTX32230), rabbit-anti-BDNF (GTX132621) (all from
GeneTex, Irvine, CA, USA); rabbit-anti-TH (ab152) and mouse-anti-
ERRγ (ab150539) (both from Abcam, Cambridge, United Kingdom);
rabbit-anti-CREB (#9197), rabbit-anti-p-Akt (#9271 L), rabbit-anti-p38
(#9212S), rabbit-anti-p-p38 (9211S), rabbit-anti-JNK (9252S), rabbit-
2.3. Rat microsomal stability
Rat liver microsome was obtained from BD Biosciences (San Jose,
CA, USA). The reaction mixture (500 μL) consisted of rat liver micro-
somal protein (0.5 mg /mL) and an NADPH regenerating system (1.3
mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-
phosphate dehydrogenase, and 3.3 mM magnesium chloride) in 100
mM potassium phosphate buffered saline (pH 7.4). The mixture was pre-
incubated in a water bath at 37 ◦C for 5 min, and the compound (HPB2
or GSK4716) was added to a final concentration of 2
L) of the mixture were sampled at 0, 5, 15, and 30 min after initiation of
the reaction. Immediately after collection, a stock solution (200 L ice-
chilled acetonitrile containing 250 ng/mL of the compound (HPB2 or
μM. Aliquots (50
μ
μ
2