Protection of primary dopaminergic midbrain neurons by GPR139 agonists supports different mechanisms of MPP+ and rotenone toxicity

Article abstract of DOI:10.3389/fncel.2016.00164

The G-protein coupled receptor 139 (GPR139) is expressed specifically in the brain in areas of relevance for motor control. GPR139 function and signal transduction pathways are elusive, and results in the literature are even contradictory. Here, we examined the potential neuroprotective effect of GPR139 agonism in primary culture models of dopaminergic (DA) neuronal degeneration. We find that in vitro GPR139 agonists protected primary mesencephalic DA neurons against 1-methyl-4-phenylpyridinium (MPP⁺)-mediated degeneration. Protection was concentration-dependent and could be blocked by a GPR139 antagonist. However, the protection of DA neurons was not found against rotenone or 6-hydroxydopamine (6-OHDA) mediated degeneration. Our results support differential mechanisms of toxicity for those substances commonly used in Parkinson’s disease (PD) models and potential for GPR139 agonists in neuroprotection.

Full text of DOI:10.3389/fncel.2016.00164

ORIGINAL RESEARCH
published: 28 June 2016
doi: 10.3389/fncel.2016.00164
Protection of Primary Dopaminergic
Midbrain Neurons by GPR139
Agonists Supports Different
+
Mechanisms of MPP and Rotenone
Toxicity
Kirsten Bayer Andersen , Jens Leander Johansen , Morten Hentzer ,
1
1
2
3
1
Garrick Paul Smith , and Gunnar P. H. Dietz *
1
2
Department of Neurodegeneration, H. Lundbeck A/S, Valby, Denmark , Department of Molecular Screening, H. Lundbeck
3
A/S, Valby, Denmark , Department of Discovery Chemistry 2, H. Lundbeck A/S, Valby, Denmark
The G-protein coupled receptor 139 (GPR139) is expressed specifically in the brain in
areas of relevance for motor control. GPR139 function and signal transduction pathways
are elusive, and results in the literature are even contradictory. Here, we examined
the potential neuroprotective effect of GPR139 agonism in primary culture models
of dopaminergic (DA) neuronal degeneration. We find that in vitro GPR139 agonists
protected primary mesencephalic DA neurons against 1-methyl-4-phenylpyridinium
+
(
MPP )-mediated degeneration. Protection was concentration-dependent and could
be blocked by a GPR139 antagonist. However, the protection of DA neurons was
not found against rotenone or 6-hydroxydopamine (6-OHDA) mediated degeneration.
Our results support differential mechanisms of toxicity for those substances commonly
used in Parkinson’s disease (PD) models and potential for GPR139 agonists in
neuroprotection.
Edited by:
Dirk M. Hermann,
University Hospital Essen, Germany
Keywords: Parkinson’s disease model,
neuroprotection, cell-based assay, neurotoxin, apoptosis
G protein-coupled receptor, neurodegeneration, drug screening,
Reviewed by:
Ertugrul Kilic,
Istanbul Medipol University, Turkey
Oleh Khalimonchuk,
University of Nebraska-Lincoln, USA
INTRODUCTION
*
Correspondence:
Gunnar P. H. Dietz
gdietz@gwdg.de
The G protein-coupled receptor (GPCR) superfamily is the largest group of cell surface
receptors. An estimated 36% of drugs approved by the United States Food and Drug
Administration during the last three decades target GPCRs, making them the most common
drug target (Rask-Andersen et al., 2011). Many GPCRs have been cloned without knowledge
of their function and ligands (Davenport et al., 2013) and are likely to cover a number
of future drug targets. One of these is the G protein coupled-receptor 139 (GPR139).
It was found to belong to the class A GPCRs and to be highly conserved through
different species including human, mouse, rat, chicken, fugu and zebrafish (Gloriam et al., 2005).
Received: 25 February 2016
Accepted: 03 June 2016
Published: 28 June 2016
Citation:
Bayer Andersen K, Leander
Johansen J, Hentzer M, Smith GP
and Dietz GPH (2016) Protection of
Primary Dopaminergic Midbrain
Neurons by GPR139 Agonists
Supports Different Mechanisms of
Abbreviations: ADME, absorption, distribution, metabolism, and excretion; 6-OHDA, 6-hydroxydopamine; BBB,
blood-brain barrier; DMEM, Dulbecco’s Modified Eagle Medium; DA, Dopaminergic; DAT, Dopamine transporter;
DIV, Days in vitro; GPCR, G protein-coupled receptor; GPR139, G protein-coupled receptor 139; HBSS,
+
+
Hank’s Balanced Salt Solution; MPP , 1-methyl-4-phenyl-pyridinium ion; MPTP, 1-methyl-4-phenyl-1 2,3,6-
MPP and RotenoneToxicity.
Front. Cell. Neurosci. 10:164.
doi: 10.3389/fncel.2016.00164
tetrahydropyridine; OCR, Oxygen consumption rate; PD, Parkinson’s disease; P/S, Penicillin/Streptomycin Solution;
SN, Substantia nigra; SNpc, Substantia nigra pars compacta; TH, Tyrosine hydroxylase; TMS, tetramethylsilane.
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Bayer Andersen et al.
GPR139 Agonists in Parkinson Model
Recently, we have identified L-Trp and L-Phe as the first
potential endogenous agonists of GPR139 (Isberg et al., 2014)
which was subsequently confirmed by researchers at Jannsen
used toxins used in PD models. We assessed toxin resistance
of primary DA cells pre-treated with the GPR139 agonists, and
whether protection by GPR139 signaling could be blocked by co-
incubation with the antagonist.
(Liu et al., 2015). In humans and mice, Gpr139 is expressed
specifically in distinct areas of the CNS, including the lateral
aspect of the striatum (Matsuo et al., 2005; Süsens et al., 2006).
Moreover, preliminary analysis suggested that Gpr139 knockout
mice display deficits in locomotion, balance and sensorimotor
tasks (Murphy and Croll-Kalish, 2004). The expression pattern
of GPR139 and the initial phenotypic analysis of the loss-
of-function are consistent with a role in the modification of
locomotor activity. Recently, while this study was in progress,
it was found that GPR139 agonists reduce locomotor activity in
rats (Liu et al., 2015). Variations in the Gpr139 locus have been
linked to inattention (Ebejer et al., 2013) in ADHD patients and
to schizophrenia (Castellani et al., 2014).
MATERIALS AND METHODS
Compounds
Compound 1
The GPR139 agonist compound 1, 2-(3, 5-Dimethoxybenzoyl)-
N-(1-naphthyl)-hydrazinecarboxamide, with an EC50 of
3
9 nM has been described earlier (Shi et al., 2011). EC50
and IC50 for all compounds were determined as described
Shi et al., 2011).
(
NMR and MS for the Preparation of
Parkinson’s disease (PD) is the second-most prevalent
neurodegenerative disease (de Lau and Breteler, 2006). The Compound 2 and 3
1
etiology of PD is still unknown in most cases, but the
characteristic motor symptoms of PD are primarily due to
the loss of neurons of the nigrostriatal dopaminergic (DA)
pathway. Treatment of PD at present is symptomatic, induces
side effects and does not stop disease progression. There is thus
a huge need for innovative and new treatment strategies (Aquino
and Fox, 2015; Bastide et al., 2015; Ossig and Reichmann, 2015).
The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyri-
dine (MPTP) is converted into the active 1-methyl-4-phenyl-
The H NMR spectra were recorded on a Bruker Avance AV
(500 MHz) with tetramethylsilane (TMS) as internal standard.
ESI-MS spectra were measured with a Thermo Finnigan
LCQ14ECAXP or a PE-Sciex API 1SO-Ex. Low-resolution EI-MS
was measured on a MAT-95 spectrometer and high resolution
ESI-MS measured with a MAT-77 spectrometer or using a
Bruker micro-TOF. NMR spectra were obtained using d6-DMSO
or CDCl3 as solvent. Chemical shifts are expressed as δ units
(ppm) relative to TMS as internal standard. The abbreviations
s, d, t, m and br refer to singlet, doublet, triplet, multiplet and
broad signal.
+
pyridinium ion (MPP ) in the brain (Przedborski and Vila, 2003)
and can produce similar biochemical and neuropathological
defects as observed in PD patients (Langston et al., 1984). These
include the progressive loss of DA neurons in the substantia Preparation of (2-Naphthalen-1-yl-acetylamino)-
nigra pars compacta (SNpc) and the decrease of striatal Acetic Acid Ethyl Ester (Figure 1)
dopamine levels. Therefore, it is one of the most widely used
experimental models for sporadic PD (Przedborski and Vila,
1-Naphthaleneacetic acid (3.03 g, 16.3 mmol) was dissolved in
dichloromethane (83.5 mL). Glycine ethyl ester hydrochloride
(2.5 g, 18 mmol) and triethyamine (4.76 mL, 34.2 mmol)
2
003). Lipophilic MPTP passes the blood-brain barrier (BBB)
◦
0
and cellular membranes. In astrocytes, monoamine oxidase B
were added and the solution cooled to 0 C under an argon
+
+
converts MPTP into MPP (Ransom et al., 1987). MPP is
taken up into DA neurons by the dopamine transporter (DAT;
Javitch et al., 1985; Mayer et al., 1986) inhibiting mitochondrial
complex I (Tipton and Singer, 1993). It promotes ATP
depletion and generation of reactive oxygen species (ROS;
Rossetti et al., 1988), which can activate apoptotic pathways
atmosphere. N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide
hydrochloride (3.43 g, 17.9 mmol) was added and the reaction
◦
was stirred for 2 h at 0 C and then at room temperature for 16 h.
The reaction was washed with saturated NaHCO (1 × 100 mL),
3
1M HCl (1 × 100 mL) and then brine (100 mL). The separated
(
Przedborski et al., 2004). Rotenone is another mitochondrial
complex 1 inhibitor applied in PD models (Betarbet et al.,
+
2
000), but unlike MPP it is lipophilic and can therefore
readily cross the cell membranes. 6-Hydroxydopamine (6-
OHDA) is taken up both by the DAT and the noradrenergic
transporter, and therefore induces cell death in both DA and
+
noradrenergic neurons (Luthman et al., 1989). Like MPP
and rotenone 6-OHDA is used for both in vitro and in vivo
models for investigations of the underlying mechanism of
PD (Ungerstedt, 1968; Sachs and Jonsson, 1975; Blesa and
Przedborski, 2014).
Recently, three agonists and an antagonist were developed
as tools to further examine GPR139 function, one of which has
been described (Shi et al., 2011). Here, we examined whether
GPR139 signaling could modify toxicity of those most commonly
FIGURE 1 | Synthesis of (2-naphthalen-1-yl-acetylamino)-acetic acid
ethyl ester. 1-Naphthaleneacetic acid was dissolved in dichloromethane.
Glycine ethyl ester hydrochloride and triethyamine were added and the
◦
solution cooled to 0 C under an argon atmosphere.
0
N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride was added
◦
and the reaction was stirred for 2 h at 0 C and then at room temperature
for 16 h.
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FIGURE 2 | Synthesis of (2-naphthalen-1-yl-acetylamino)-acetic acid.
(
2-Naphthalen-1-yl-acetylamino)-acetic acid ethyl ester was dissolved in
ethanol. Sodium hydroxide was added and the reaction was stirred for 16 h at
room temperature.
organic layer was dried (MgSO4), filtered and concentrated in
vacuo to give the desired product. Yield: 3.6 g, 81%.
1
H NMR (500 MHz, CDCl3) δ 8.0 (d, 1H), 7.91 (d, 1H), 7.84
(
4
d, 1H), 7.59–7.50 (m, 2H), 7.50–7.43 (m, 2H), 5.53 (s, 1H),
.14–4.07 (m, 4H), 3.93 (d, 2H), 1.19 (t, 3H).
Preparation of (2-Naphthalen-1-yl-acetylamino)-
Acetic Acid (Figure 2)
(
2-Naphthalen-1-yl-acetylamino)-acetic acid ethyl ester (4.0 g,
0 mmol) was dissolved in ethanol (100 mL). Sodium hydroxide
2 M, 22.1 mL) was added and the reaction was stirred for 16 h
1
(
at room temperature. The reaction mixture was concentrated
in vacuo and 2M HCl added until the mixture was acidic. The
product precipitated and was filtered. The solid was washed with
water and ether and dried in a vaccuum. Yield: 2.85 g, 80%.
FIGURE 3 | Synthesis of Compound 2. (A) ((1-Naphthylacetyl)Amino)acetic
acid was dissolved in dichloromethane and N, N-diisopropylethylamine at
◦
room temperature and cooled to 0 C. To the solution was added
(
3-dimethylamino)pyrrolidine and N, N’-dicyclohexylcarbodiimide. (B) 1H NMR
1
H NMR (500 MHz, DMSO-d6) δ 8.51 (d, 1H), 8.09 (d, 1H),
spectrum of compound 2, with specifications provided in “Materials and
Methods” Section.
7
.91 (d, 1H), 7.80 (br s, 1H), 7.51)br s, 2H), 7.44 (br s, 1H), 3.10
(
s, 2H), 3.78 (s, 2H).
Preparation of Compound 3:
N-[(2-Methoxy-ethyl)-Methyl-carbamoyl]-methyl-2-
Naphthalen-1-yl-acetamide (Figure 4)
Preparation of Compound 2:
N-[2-(3-Dimethylamino-pyrrolidin-1-yl)-2-oxo-ethyl]-
2
-Naphthalen-1-yl-acetamide (Figure 3)
(2-Naphthalen-1-yl-acetylamino) acetic acid (400 mg, 2 mmol),
N-(2-Methoxyethyl)methylamine (138 mg, 1.55 mmol) and
triethylamine (0.433 mL, 3.11 mmol) were dissolved in
(
2-Naphthalen-1-yl-acetylamino) acetic acid (600 mg,
2
.46 mmol) was dissolved in dichloromethane (75 mL) and
◦
N,N-diisopropylethylamine (1.1 mL, 6.2 mmol) under an
nitrogen atmosphere at room temperature then and cooled to
dichloromethane (10 mL) and cooled to 0 C. N-(3-Dimethyl
0
aminopropyl)-N -ethylcarbodiimide hydrochloride (447 mg,
◦
◦
0
C. To the solution was added (3-dimethylamino)pyrrolidine
2.33 mmol) was added and the reaction was stirred at 0 C for
0
(
235 mg, 2.06 mmol) and N,N -dicyclohexylcarbodiimide
848 mg, 4.1 mmol). The mixture was stirred overnight at
2 h and then for 16 h at room temperature. The reaction was
(
washed with saturated sodium bicarbonate, 1M HCl and brine.
room temperature. The reaction mixture was washed twice
with aq. NaOH (1M), brine (50 mL), dried with MgSO4,
filtered and concentrated in vacuo. The crude product
was purified by flash chromatography using heptane/ethyl
acetate/trimethylamine/methanol (1:1:0.05:0.1). The purified
product was isolated as a solid. Yield: 520 mg, 62%.
The organic layer was dried with MgSO and the compound was
4
concentrated in vacuo. The crude product was purified by flash
chromatography eluting with heptane/ethyl acetate 1:1. Yield:
0.33 g, 68%.
+
1
LC-MS (M + H) found at 315.3. H NMR (500 MHz, CDCl )
δ 8.00 (d, 1H), 7.83 (d, 1H), 7.78 (m, 1H), 7.50–7.44 (m, 2H),
3
+
1
LC-MS [M + H] found at 340.3. H NMR (500 MHz,
7.43 (d, 2H), 6.61 (d, 1H), 4.05–4.03 (br s, 2H), 4.02 (d, 1H), 3.94
(d, 1H), 3.46–3.34 (m, 4H), 3.30–3.26 (m, 1H), 3.24 (d, 2H), 2.89
(s, 2H), 2.84 (s, 1H).
The surrogate GPR139 agonist compound 3 had an EC50 of
850 nM.
DMSO- d6) δ 8.24 (1H, dd), 8.12 (1H, dd), 7.93 (m, 1H), 7.83
(
(
(
(
(
d, 1H), 7.56–7.43 (m, 3H), 4.00 (s, 2H), 3.88 (m, 2H), 3.65–3.5
m, 2H), 3.36 (s, 4H), 3.18 (m, 0.5H), 3.11 (m, 0.5H), 3.01–2.95
m, 2H), 2.67 (dd, 0.5H), 2.56 (dd, 0.5H), 2.14 (d, 1H), 2.05
m, 0.5H), 1.98, (m, 0.5H), 1.72 (m, 0.5H), 1.57 (m, 0.5H), 0.98
m, 1H).
Compound 4
The GPR139 surrogate agonist compound 2 had an EC50 of
The
surrogate
GPR139
antagonist
compound
4,
5
30 nM.
1-(4-Fluoro-phenyl)-2-methyl-3-(2,2,2-trichloro-ethyl)-1,5,6,7-
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GPR139 Agonists in Parkinson Model
FIGURE 4 | Synthesis of compound 3. (A) [(1-Naphthylacetyl)amino]acetic
acid, N-(2-Methoxyethyl)methylamine and triethylamine were dissolved in
◦
0
dichloromethane and cooled to 0 C. N-(3-Dimethylaminopropyl)-N -ethyl-
◦
carbodiimide hydrochloride was added and the reaction was stirred at 0 C
and then at room temperature. (B) H NMR spectrum of compound 3, with
1
specifications provided in “Materials and Methods” Section.
tetrahydro-indol-4-one, is commercially available. It was
purchased from Specs (Zoetermeer, Netherlands). It had an IC50
of 7.4 µM, and was therefore applied at 10 µM in cell culture
experiments.
None of the compounds were useful in vivo tools due
to their unfavorable ADME properties including low whole
brain exposure and brain/plasma ratio (Shi et al., 2011). Cross
reactivity with the DAT or the norepinephrine transporter was
determined as described (Shi et al., 2011).
FIGURE 5 | Additional characterization of GPR139 compounds using
kinetic fluorescence measurements and concentration-response
determination in calcium mobilization assays. (A–D) Examples for the
kinetic of relative fluorescence units (RFU) for compound 1 and 4 and related
controls, involving a two-step addition protocol. (A) Cellular fluorescence
response to addition of buffer (approximately at time 0:00 min) and a second
addition of buffer at 3:20 min. (B) Cellular fluorescence response to addition of
GPR139 Calcium Mobilization Assay
All compounds were further characterized using GPR139-based
2+
Ca mobilization assays (Figure 5).
Standard molecular cloning techniques were used to generate
Chinese hamster ovary (CHO-K1) cells stably expressing
the human GPR139 receptor. The cell line was grown in
Ham’s F-12K (Kaighn’s) medium (Gibco 21127), 10% FBS
2+
2+
the Ca ionophore and reference compound for complete release of Ca
from intracellular stores, ionomycin. (C) Cellular response to addition of 10 µM
compound 1 at time 0:00. (D) Cellular fluorescence in response to antagonist
compound 4 (at 0:00, 50 µM) and addition of agonist compound 1 at EC85
concentration. (E–H) Normalized response (stimulation by agonist) for
compound 1 (E) compound 2 (F), and compound 3 (G) in G-protein coupled
(
Gibco 10091–155), 1% Sodium Pyruvate (Gibco 11360),
.5 mg/ml G418 (Gibco 11811–064), 1% Penicillin Streptavidin
Gibco 15140–122). Cells expressing GPR139 were plated
0
2+
receptor 139 (GPR139) Ca mobilization assay. (H) Concentration-response
(
(inhibition by antagonist) for compound 4.
in growth medium (modified to contain 5% FBS, 0.5%
Penicillin/Streptavidin and 1 × ITS-X(Gibco #51500–056)) at a
density of 10,000 cells/well (30 µl) in clear-bottomed, poly-D-
lysine coated 384-well plates (ArcticWhite LLC, Bethlehem, PA,
HEPES, pH 7.4). The cells were incubated with a Ca2 -sensitive
fluorescent dye, Calcium4 (Molecular Devices Inc., Sunnyvale,
CA, USA) with 2.5 mM Probenecid (Sigma, St. Louis, MO,
+
◦
◦
USA) and grown for 24 h at 37 C in the presence of 5% CO2.
USA) for 50 min at 37 C and followed by 10 min at room
temperature. Calcium flux was measured using a Hamamatsu
FDSS7000 imaging-based plate reader (Hamamatsu Photonics)
Before assaying, the cells were washed with assay buffer (Hanks’
2+
2+
balanced salt solution with Ca and Mg containing 20 mM
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Bayer Andersen et al.
GPR139 Agonists in Parkinson Model
using 480 nm excitation light and emitted fluorescent light
passed through a 525 nm emission filter and detected by a
CCD camera. Test compounds were diluted in assay buffer
from 2 or 10 mM stock solutions in 100% DMSO to give a
3
× concentrated stock. Compounds were added to cells and
fluorescence measured at 1 Hz starting just prior to compound
addition. The fluorescence readout was calculated as max-
min response, i.e., maximum fluorescence reading after and
before liquid addition. The fluorescence max-min data were
normalized to yield responses for no stimulation (buffer) and full
stimulation (5 uM compound 1) of 0% and 100% stimulation,
respectively. Antagonism was examined as inhibition of agonist-
induced stimulation via a subsequent 2nd addition step of
compound 1 at EC85 concentration (appx at time 3:20 min). The
fluorescence max-min data were normalized to yield responses
for no stimulation (buffer) and EC85 stimulation of 100% and 0%
inhibition, respectively. Concentration-response data were fitted
to the four-parameter logistic equation to estimate compound
potency (EC50 or IC50) and efficacy (Emax or Imax) (Motulsky and
Christopoulos, 2003).
Primary DA Midbrain Neuron Culture and Toxicity
Assays
FIGURE 6 | Tyrosine-hydroxylase positive neurons in primary
Experiments were conducted in accordance with the ethical
guidelines of H. Lundbeck A/S and the Danish legislation
of animal use for scientific procedures. Cell culture was
basically performed as described (Nagel et al., 2008), with few
modifications. Briefly, the mesencephalon floor was dissected
from embryonic day (E) 13 mice, tissue pieces collected in
HBBS, transferred to 0.1% trypsin, 0.05% DNAse (Sigma-Aldrich
mesencephalic cultures. Pixels with an intensity above a set threshold that
the image analysis software detects as TH-positive are shown in green; nuclei
of TH-positive cells detected by the software are marked in light blue. Space
bar: 50 µM.
Figures 4, 6. Results were normalized against control condition
and compared. Experiments were only included in the evaluation
if at least 30% cell death was induced at the highest toxin
concentration compared to non-treated control wells. To ensure
that only healthy primary cultures were assessed, we furthermore
excluded experiments with total cell counts below Mean
minus standard deviation (SD) of the summarized experiments.
Statistical significance of the differences between the control
condition and agonist treatment was investigated by the unpaired
t test at the 5% level using GraphPad online software.
◦
DN25–1G) and incubated at 37 C in a humidified atmosphere
with 5% CO2 for 20 min; washed in DNAse, homogenized and
centrifuged at 200× g, resuspended in medium (DMEM (Gibco
4
1965–039), 10% FCS, 1% P/S (Gibco, 15140–122), l.5% HEPES
(
Gibco, 15630–056), 1% Sodium Pyruvate (Gibco, 11360–039))
and plated on poly-L-lysine coated 96-well dishes. The medium
was switched to neurobasal medium after 90 min (neurobasal
(
Gibco 21103–049) with 1% P/S, 2% B27 (Gibco, 17504–044),
1
.25% 0.5 mM L-glutamine (Gibco, 25030–024)). After 6 days
in vitro (DIV), cultures were treated with 1 µM of three different
Real-time Quantitative PCR
GPR139 agonists and in some experiments, concomitantly, with
RNA was isolated using the RNAeasy kit from Qiagen, Valencia,
CA, USA. Briefly, RNA was reverse transcribed and detection
of PCR gene-fragments was carried out on a MJ Research light
1
0 µM of the antagonist for1 h, before exposing them to 0–1 µM
+
MPP , 0–50 µM 6-OHDA or 0–100 nM rotenone for 24 h. For
+
the MPP treated cultures, it was also tested whether the effect of
cycler by SYBR detection. The Q-PCR results were analyzed
the GPR139 agonists could be blocked by concomitantly applying
−∆∆CT
by the 2
method as earlier described (Pfaffl, 2001) using
1
0 µM GPR139 antagonist (IC50 3 µM). Subsequently, cells were
cyclophilin A as a control reference.
fixed in 4% PFA/PBS for 10 min, washed with PBS, incubated
in 0.5% BSA/0.1% Triton-X/5% porcine serum for 20 min,
and incubated for 24 h in rabbit anti-tyrosine hydroxylase
antibodies (1:1000 in PBS, Millipore AB152); washed 2 × 5 min
in PBS. Secondary antibodies (Alexa 488 rabbit) were applied
RESULTS
Expression of Gpr139 in Primary DA
1
2
:200 in PBS with 0.2 µg/ml Hoechst dye for 1 h and washed Midbrain Neurons
× 5 min in PBS. One drop of DAKO fluorescent mounting
We used quantitative PCR to determine relative expression levels
of Gpr139 in primary midbrain neuron cultures. C(t) values using
two different primer pairs for Gpr139 were in the same range as
the C(t) value for tyrosine hydroxylase, the gene characteristic
for DA neurons (Figure 7A). Gpr139 expression was about
◦
medium were applied into each well. Plates were stored at 4 C
until tyrosine-hydroxylase-positive (TH-positive) neurons were
r
r
counted using a Thermo Scientific Cellomics ArrayScan VTI
HCS Reader. An example image of such cell culture is provided in
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GPR139 Agonists in Parkinson Model
FIGURE 7 | Gpr139 is expressed in primary midbrain cultures. Results from quantitative polymerase chain reaction (PCR) showing c(t) values for expression of
(
(
A) the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH); Cyclophilin A; Gpr139 detected by two different primer pairs; and TH.
B) Expression of Gpr139 in the mouse fibroblast cell line NIH 3T3 and the mouse neuroblastoma cell line N2a and primary midbrain cultures (VM), normalized to
Cyclophilin A. Shown are the mean values of the experiment run in triplicates. Error bars represent standard deviations of the mean.
two orders of magnitude higher in primary midbrain cultures,
compared to expression in a mouse fibroblast cell line, or a
neuroblastoma cell line (Figure 7B).
the cultured DA midbrain neurons with rotenone, which, like
+
MPP , is a mitochondrial complex I inhibitor. While rotenone
did induce a dose-dependent cell death in the TH-positive
neurons, no rescue was seen in the agonist treated sub-
population (Figure 11).
Protection Against Toxin-Induced Cell
Death
Agonist-Dopamine Transporter (DAT)
To examine whether GPR139 agonists protect against toxin- Interaction
+
induced DA cell death, we first treated cultured DA midbrain
As both 6-OHDA and MPP are taken up via the DAT, it was
not likely that the agonists mediated their protection against
+
neurons with the GPR139 agonists and MPP . Subsequently, we
+
determined survival of TH-positive neurons. We found that three
different agonists dose-dependently and substantially protect
MPP by merely blocking the DAT. However, to further exclude
+
the possibility that the protection against MPP by the agonist
compound 3 is mediated by blocking MPP uptake, rather than
+
+
primary DA neurons against MPP toxicity: between 40.5%
(
compound 2) and 42.8% (compound 3) of the cells killed by
signaling through GPR139, DA and noradrenaline uptake in
the presence of the agonist was tested. At 10 µM compound 3
inhibited DA uptake only 13% and norepinephrine uptake only
−13%, suggesting that the protective effect observed with the
agonist is likely not mediated by directly acting on the DAT.
Ten micrometres (10 µM) compound 1 inhibited binding to
the DAT or the norepinephrine transporter by 4% or −8%,
respectively (Shi et al., 2011) compound 2 inhibited binding
to the DAT or the norepinephrine transporter 2% or −4%,
respectively.
We next sought to examine whether the agonists would also
provide neuroprotection in vivo. However, we found that ADME
properties of the compounds were not favorable to provide
sufficient brain exposure and receptor occupancy, neither by oral
application, nor by subcutaneous delivery via osmotic pumps
(data not shown).
+
1
µM MPP were rescued by previous incubation with 1 µM
of either compound (Figure 8).
To determine whether the observed protection acted
specifically on GPR139, we tested whether the effect was
reversible by a GPR139 antagonist. While the antagonist alone
did not have an effect on DA cell survival, the antagonist blocked
the protective effect of the agonists (Figure 9).
To determine whether the GPR139 agonists would also
protect against other toxins affecting DA neurons, we
examined the effect of the agonists on 6-OHDA toxicity.
We found that GPR139 agonists did not protect cultured DA
midbrain neurons against extended culture periods or 6-OHDA
(
Figure 10).
To examine whether the agonist-induced protection likely
was mediated via mitochondrial complex I inhibition, we treated
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GPR139 Agonists in Parkinson Model
FIGURE 8 | Three different GPR139 surrogate agonists protect primary
dopaminergic (DA) midbrain neurons against 1-methyl-4-phenyl-
pyridinium ion MPP toxicity. Neuronal midbrain cultures were pretreated
FIGURE 10 | GPR139 agonists do not protect cultured DA midbrain
neurons against 6-OHDA toxicity. Neuronal midbrain cultures were
pretreated with 1 µM of either one of the agonists compound 1 ( ), compound
+
with 1 µM of either one of the agonists compound 1 ( ), compound 2 ( ), or
2
( ), or compound 3 ( ), or vehicle ( ) for 1 h, followed by treatment with the
compound 3 ( ), or vehicle ( ) for 1 h, followed by treatment with the indicated
indicated concentrations of 6-hydroxydopamine (6-OHDA) for 24 h.
TH-positive neurons were counted and normalized to control numbers.
+
concentrations of MPP for 24 h. TH-positive neurons were counted and
+
normalized to numbers under control conditions. At 1 µM MPP , protection
∗
by all three different agonists was significant ( p ≤ 0.05). Each data point is
+
calculated from 12 (0 µM and 1 µM MPP concentrations); 4 (0.125 µM); 7
(
0.25 µM); or 8 (0.5 µM) independent measurements.
DISCUSSION
We demonstrate that three GPR139 agonists dose-dependently
To confirm the motor deficits earlier described in Gpr139
+
protect primary DA neurons against MPP toxicity. When
KO mice, the mice underwent behavioral testing on a rotarod
with a fixed and increasing speed and a balance beam paradigm.
We could not detect any differences among Gpr139 KO mice,
heterozygous mice, or wt mice (data not shown). This may,
however, be due to the fact that the mice were on a non-congenic
background different from the ones used in earlier studies
treating cultured DA midbrain neurons with rotenone or
6
-OHDA, we also observed dose-dependent cell death; however,
GPR139 agonist treatment did not rescue the neurons. Moreover,
no protection was demonstrated against prolonged culture
periods, suggesting that GPR139 agonism does not enhance
general cellular viability and resistance against apoptotic stimuli.
Previously, not peer-reviewed work suggested that GPR139
KO mice display a deficit in motor performance (Murphy and
Croll-Kalish, 2004). However, in pilot experiments, we could not
confirm those deficits using the rotarod and the balance beam
test. That discrepancy might be due to a different and variable
background of the mice examined compared to the mice in the
earlier study.
(
Murphy and Croll-Kalish, 2004).
Work by Song et al. (2012) has previously reported that the
bibenzyl compound Chrysotoxine could antagonize the toxicity
+
of MPP , but not rotenone in SH-SY5Y cells. However, the
+
authors explain that the MPP protection is, at least partly,
due to inhibition of the DAT (Song et al., 2012). In our
+
study the protective effect of the agonists seen in the MPP
treated cultures is unlikely to be due to DAT inhibition.
First, 6-OHDA is also partly taken up via the DAT and no
protection of the agonists was seen when cultures were treated
with 6-OHDA. Next, agonist compound 1 and compound 2
did not show cross-reactivity with the DAT. It cannot be
entirely ruled out that compound 3 does interfere with the
DAT; however, considering the very similar effect of the
+
FIGURE 9 | Protection of DA midbrain neurons against MPP toxicity
by a GPR139 agonist is dose dependent and can be blocked by a
GPR139 antagonist. Primary midbrain cultures were pretreated with the
indicated amount of GPR139 agonist compound 3 and with either vehicle ( ),
+
+
or 1 µM MPP
( ); or MPP with 10 µM of the antagonist compound 4 ( ),
or with the antagonist compound 4 alone ( ). The number of TH-positive
neurons was determined 24 h later and normalized to control conditions.
+
three agonist on MPP toxicity, that mechanism is not very
likely.
Statistical significance compared to vehicle treated cells is indicated
We further examined the specificity of the agonist protection
∗
(
p < 0.05).
+
against MPP toxicity by showing that a GPR139 antagonist
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Bayer Andersen et al.
GPR139 Agonists in Parkinson Model
rotenone would likely induce cell death in the entire cell
population it becomes exposed to. Indeed, both in vivo and
in vitro rotenone has been found to result in unspecific DA
and non-DA neuronal cell death in some studies (Nakamura
et al., 2000; Tieu, 2011). However, using different experimental
approaches, DA neurons are particularly vulnerable (Ferrante
et al., 1997; Betarbet et al., 2000; Bywood and Johnson,
2
003; Kweon et al., 2004; Radad et al., 2008). It has been
+
suggested that chronic exposure to low MPP (Nakamura
et al., 2000) or rotenone (Kweon et al., 2004) concentrations
is selectively more toxic to DA neurons, whereas acutely
administered high concentrations cause less discriminative cell
death. DA neurons are more vulnerable to mitochondrial energy
disruption than non-DA neurons (Kweon et al., 2004), which
might explain why DA neurons can be selectively killed by
rotenone.
FIGURE 11 | GPR139 agonists do not protect cultured DA midbrain
neurons against rotenone toxicity. Mesencephalic cultures were treated
with 1 µM of either one of the GPR139 agonists compound 1 ( ), compound
While we observed an average DA neuronal cell death in the
primary cultures of 60% after 24 h with 100 nM rotenone and
4
+
+
0% with 1 µM MPP , in our experience it also takes nM MPP
2
0
( ), or compound 3 ( ), or vehicle ( ) for 1 h before exposing them to
–100 nM of rotenone. Results are average of ± SEM (n = 4 independent
concentrations to achieve 50% cell death in the neuroblastoma
SH-SY5Y cell line (not shown). Nevertheless, as those cells
express DA markers, they also provide a useful model system to
study the mechanism of toxicity induced by substances used in
PD models. Giordano et al. (2012) found a number of differences
experiments). TH-positive neurons were counted and normalized to control
numbers (100%). At a given rotenone concentration, none of the values
among the treatment groups were significantly different from one another.
+
could block the protection, while the antagonist itself was not
toxic to DA neuron cultures. Thus, together with the fact that
three different agonists showed similar effects, and the low cross-
reactivity of the compounds in a broad pharmacology panel
screen, assaying the ability to displace radioligand binding to the
assayed targets (Shi et al., 2011), it is likely that the protection we
observed is mediated through GPR139.
in the effects of MPP and rotenone on bioenergetics and cell
death in differentiated SH-SY5Y neurons. 50% cell death was
obtained after 24 h incubation with 5 nM rotenone, 5 mM
+
MPP or 100 µM 6-OHDA. Increasing doses of rotenone
resulted in significant cell death and caspase 3 activation.
Rotenone immediately inhibited the mitochondrial basal oxygen
consumption rate (OCR) with a resulting decrease of ATP-linked
OCR, reserve capacity and a stimulation of glycolysis. With high
Our data complement previous findings on the mechanism
of GPR139 signal transduction. Most studies, including recent
ones from our own group (Matsuo et al., 2005; Süsens et al.,
+
doses of MPP nearly eliminating basal and ATP-linked OCR,
less pronounced cell death was seen compared to that induced by
rotenone. Cytotoxic 6-OHDA doses had much lower impact on
bioenergetics functions and thus Giordano et al. (2012) suggests
that its toxic effect is probably independent of these (Giordano
et al., 2012).
2
006; Shi et al., 2011; Isberg et al., 2014; Dvorak et al., 2015;
Liu et al., 2015) suggest that the receptor mobilizes intracellular
calcium, which can be blocked with the Gq inhibitors YM-254890
(
Matsuo et al., 2005) and UBO-QIC (Isberg et al., 2014),
which is characteristic of Gq pathway activation. Moreover,
While the inhibition of the mitochondrial complex I plays
+
as shown in Figure 5, the GPR139 agonists used in this
a substantial role in both the toxicity of MPP and rotenone
2+
2+
study also activate Ca mobilization. Downstream of Ca
(Sherer et al., 2003; Richardson et al., 2007), several studies have
suggested that it cannot account for the entire toxic effect seen
in DA neurons (Nakamura et al., 2000; Kweon et al., 2004;
Choi et al., 2008). It is thus possible that GPR139 agonists
have different effects in the two model systems due to the
activation, there are a ‘‘myriad’’ (Dunn and Ferguson, 2015)
of mechanisms that control GPCR signaling and trafficking.
Which of those signal transduction pathways are relevant upon
GPR139 activation remains to be investigated. There are also
reports on additional activation of the Gi (Süsens et al., 2006)
or the Gs (Hu et al., 2009) pathways. It can thus not be ruled
out that GPR139 might also activate other pathways under
certain circumstances, further indicating the complexity of those
downstream events.
+
differential effects of MPP and rotenone on mitochondrial
complex I inhibition, and on the other hand by acting on
pathways independent of those. Whether GPR139 activation e.g.,
inhibits the apoptotic pathway, for instance by interfering with
molecules of the Bcl-2 family, or by stabilizing mitochondrial
integrity, etc., should be addressed by future experiments.
A few previous studies have shown differences in the effects
+
+
of MPP and rotenone. While both are mitochondrial complex I
Although neuroprotection against MPP , rotenone, or
inhibitors, rotenone is more potent (Higgins and Greenamyre,
6-OHDA often does not translate into the identification of
PD treatment targets or drug candidates, it does go beyond
merely providing a model system to study the effects of specific
elimination of DA neurons. Mitochondrial dysfunction is likely
to also play an important role in PD (Schapira and Gegg, 2011).
1
996; Mizuno et al., 1987). Rotenone is lipophilic, and it
+
can thus readily cross the cell membrane, whereas MPP
depends on the DAT for transport into cells (Javitch et al.,
1
985). One could speculate that due to its easy entry to cells
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Bayer Andersen et al.
GPR139 Agonists in Parkinson Model
Moreover, exposure to rotenone and other pesticides in farming
communities substantially increases the incidence of PD (Tanner
et al., 2011; Kamel et al., 2014). Thus, understanding the
mechanisms of toxin-induced DAergic neuronal death could also
contribute to a deeper understanding of some aspects of disease
mechanisms.
KO mouse on a congenic background may render additional
information on GPR139 function.
AUTHOR CONTRIBUTIONS
All authors finally approved the version to be published and
agreed to be accountable for all aspects of the work. KBA
acquired and analyzed the data on rotenone neuroprotection as
well as the agonist in vivo data, and wrote the initial draft of
the manuscript. JLJ acquired and analyzed the data on Gpr139
expression, analyzed that data, and wrote the corresponding
section of the work. MH acquired and analyzed the data on
compound screening, cross-reactivity and EC determination,
+
In conclusion, the difference in protection against MPP and
rotenone might be explained by a more pronounced bioenergetic
effect of rotenone toxicity, and other pathways affected by
the toxins beyond mitochondrial complex I inhibition. The
+
protective effect of 3 different agonists against MPP could be
reversed by a GPR139 antagonist. Together with the missing
protection against 6-OHDA this points towards a specific
protective effect of the agonists mediated through GPR139. Our
results further substantiate differences in the effect of three of the
most commonly used toxins in PD models.
50
and wrote the corresponding sections of the manuscript. GPS did
the conception to generate, analyze and identify the compounds,
and wrote the corresponding section of the manuscript. GPHD
initiated and designed the concept of the work; was involved in
As described earlier by Shi et al. (2011) agonist compound 1
presented in this study does not hold the ADME properties
necessary for in vivo testing. The same applies to agonists
compound 2 and compound 3. While this manuscript was in
preparation, a selective, high-affinity GPR139 small molecule
agonist with favorable ADME properties and high brain exposure
was developed (Dvorak et al., 2015). It was found that that agonist
reduced rat motor activity in vivo (Liu et al., 2015). To validate
whether activation of GPR139 in vivo has a protective effect on
DA neurons that GPR139 agonist could be used in MPTP studies
in mice to evaluate rescue of DA neurons in agonist treated
animals. Furthermore, detailed characterization of the GPR139
+
the data generation on MPP and 6-OHDA toxicity and revised
and wrote the final versions of the manuscript.
ACKNOWLEDGMENTS
We thank Lone Lind Hansen (Lundbeck A/S) for expert
technical assistance and Hans Bräuner-Osborne (University of
Copenhagen) and Jan Egebjerg (Lundbeck A/S) for many useful
discussions and comments on the manuscript. Funding was
provided from the Danish Agency for Science, Technology and
Innovation (KBA).
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Conflict of Interest Statement: While this work was in progress, all authors were
employed and paid by the pharmaceutical company H. Lundbeck A/S in Valby,
Denmark, which focuses on research, development, production, marketing and
sale of medication for the treatment of psychiatric and neurological diseases.
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