Journal of Molecular Neuroscience

https://doi.org/10.1007/s12031-018-1181-4

Antioxidant and Anti-Apoptotic Activity of Octadecaneuropeptide

Against 6-OHDA Toxicity in Cultured Rat Astrocytes
Hadhemi Kaddour 1,2,3 & Yosra Hamdi 1 & Fatma Amri 1 & Seyma Bahdoudi 1,4 & Ibtissem Bouannee 1 & Jérôme Leprince 4,5 &
Sami Zekri 6 & Hubert Vaudry 4,5 & Marie-Christine Tonon 4 & David Vaudry 4,5 & Mohamed Amri 1 & Sana Mezghani 1 &
Olfa Masmoudi-Kouki 1

Received: 25 July 2018 / Accepted: 21 September 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Oxidative stress, associated with various neurodegenerative diseases, promotes ROS generation, impairs cellular antioxidant
defenses, and finally, triggers both neurons and astroglial cell death by apoptosis. Astrocytes specifically synthesize and release
endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN). We have previously reported that ODN
acts as a potent neuroprotective agent that prevents 6-OHDA-induced apoptotic neuronal death. The purpose of the present study
was to investigate the potential glioprotective effect of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat
astrocytes. Incubation of astrocytes with graded concentrations of ODN (10−14 to 10−8 M) inhibited 6-OHDA-evoked cell death
in a concentration- and time-dependent manner. In addition, ODN prevented the decrease of mitochondrial activity and caspase-3
activation induced by 6-OHDA. 6-OHDA-treated cells also exhibited enhanced levels of ROS associated with a generation of
H2O2 and O2°-, and a reduction of both superoxide dismutase (SOD) and catalase (CAT) activities. Co-treatment of astrocytes
with low concentrations of ODN dose-dependently blocked 6-OHDA-evoked production of ROS and inhibition of antioxidant
enzyme activities. Concomitantly, ODN stimulated Mn-SOD, CAT, glutathione peroxidase-1, and sulfiredoxin-1 gene transcrip-
tion and rescued 6-OHDA-associated reduced expression of endogenous antioxidant enzymes. Taken together, these data
indicate that, in rat astrocytes, ODN exerts anti-apoptotic and anti-oxidative activities, and hence prevents 6-OHDA-induced
oxidative assault and cell death. ODN is thus a potential candidate to delay neuronal damages in various pathological conditions
involving oxidative neurodegeneration.

Keywords Astrocytes . Octadecaneuropeptide . Oxidative stress . Cell death . 6-hydroxydopamine . Neuroprotection

Hadhemi Kaddour and Yosra Hamdi contributed equally to this study.

Abbreviations
* Olfa Masmoudi-Kouki 6-OHDA 6-hydroxydopamine
olfa.masmoudi@fst.utm.tn CNS Central nervous system
DBI Diazepam-binding inhibitor
1
University Tunis El Manar, Faculty of Sciences of Tunis, LR18ES03, DCFH 2′,7′-dichlorodihydrofluorescein
Laboratory of Neurophysiology, Cellular Physiopathology and
JC-1 5,5″,6,6″-tetrachloro-1,1″,3,3″-
Biomelcules Valorisation, 2092 Tunis, Tunisia
2
tetraethylbenzimidazolylcarbocyanine iodide
CIRB, CNRS UMR 7241/INSERM U1050, PSL University, Labex
DHE Dihydroethidium
MemoLife, Collège de France, 11 place Marcelin Berthelot,
75231 Paris, France DHR123 Dihydrorhodamine 123
3 FDA Fluorescein diacetate
Imagine Institute and Center of Psychiatry and Neuroscience,
Université Paris Descartes, 102-108 rue de la Santé, Gpx Glutathione peroxidase
75014 Paris, France H2O2 Hydrogen peroxide
4
UNIROUEN, Inserm U1239, Laboratory of Neuronal and LDH Lactate dehydrogenase
Neuroendocrine Communication and Differentiation, Normandie NBT Nitrotetrazolium blue chloride
Univ, 76000 Rouen, France O2°− Superoxide anions
5
UNIROUEN, Regional Cell Imaging Platform of Normandy ODN Octadecaneuropeptide
(PRIMACEN), Normandie Univ, 76000 Rouen, France PD Parkinson’s disease
6
USCR Transmission Electron Microscopy, Faculty of Medicine, ROS Reactive oxygen species
University Tunis El Manar, Tunis, Tunisia

SOD Superoxide dismutase
Srxn Sulfiredoxin
Introduction
Astrocytes have long been considered as just providing tro-
phic support to neurons, but their importance in many func-
tions, such as neurotransmission, cell signaling, inflammation,
and synapse modulation is now well established (Dallerac and
Rouach 2016; Dzyubenko et al. 2016; Garzon et al. 2016; Liu
and Chopp 2016). Currently, the involvement of astrocyte
dysfunction in the pathophysiology of neurological disorders,
including neurodegenerative diseases and stroke, is highlight-
ed (Garzon et al. 2016; Pekny et al. 2016). Parkinson’s disease
(PD) is a neurodegenerative disorder that is primarily charac-
terized by a progressive loss of dopaminergic neurons within
the substantia nigra pars compacta (SNpc) (Michel et al. 2013;
Toulorge et al. 2016). One of the widely used models of PD
involves treatment with 6-hydroxydopamine (6-OHDA), a
neurotoxin that selectively damages catecholaminergic neu-
rons both in vivo and in vitro (Blesa and Przedborski 2014).
In vitro experiments show that the sensitivity of neurons to 6-
OHDA is mainly associated with an auto-oxidation-mediated
overproduction of reactive oxygen species (ROS), such as
hydrogen peroxide (H2O2), superoxide anion (O2°-), and hy-
droxyl radical (OH°) (Han et al. 2014; Soto-Otero et al. 2000).
The main cellular defense mechanism to cope with oxidative
stress is based on antioxidant molecules and enzymes, i.e.,
glutathione, catalase, superoxide dismutases (SODs), glutathi-
one peroxidase (Gpx), and sulfiredoxin1 (Srxn1), which can
prevent cell oxidative stress damage (Afshin-Majd et al. 2017;
Kiasalari et al. 2016; Zhou et al. 2015a), and thus play a major
neuroprotective role against the deleterious effects of 6-
OHDA. Treatment of neuroblastoma cell lines with
astrocyte-conditioned medium, significantly increases the re-
sistance of neurons to oxidative stress induced by 6-OHDA
(Murakami et al. 2018). It has been shown that the reduction
of neuronal cell death induced by astrocyte-conditioned me-
dium is associated with a stimulation of glutathione peroxi-
dase expression, an increase of glutathione levels and activa-
tion of the production of neuroprotective factors (Gardaneh
et al. 2011; Ghouili et al. 2018; Sandhu et al. 2009). These
data highlight the role of astrocyte-derived factors to maintain
cerebral antioxidant competences and to prevent neuronal
damages.
Activation of glia, which is a common pathological feature
of multiple neurodegenerative conditions (Burda et al. 2016;
Lev et al. 2013; Osborn et al. 2016), is characterized by a
series of metabolic and morphological changes of astrocytes,
and is notably observed in the substantia nigra and cerebral
cortex of human PD brains as well as in 6-OHDA-treated
animals, a model of PD (Gomide et al. 2005; Wachter et al.
J Mol Neurosci
2010). Although activated astrocytes were traditionally
thought to impede neuronal regeneration by forming glial
scars (Lev et al. 2013; Pekny et al. 2016), growing evidence
indicates that reactive astrocytes contribute to functional re-
covery after brain injuries (L’ Episcopo et al. 2010;
L’Episcopo et al. 2011). Concurrently, analysis of postmortem
PD brains has revealed a higher neuronal loss within areas
where astrocytes are less abundant (Gu et al. 2010; Mirza
et al. 2000). Moreover, it has been shown that, in rodents,
injection of 6-OHDA within the nigrostriatal pathway pro-
duces a loss of astrocytes associated with the degeneration
of dopaminergic neurons (Espinosa-Oliva et al. 2014).
Indeed, astroglial cells are a source of neurotrophic factors
that can protect remaining neurons against oxidative stress
by detoxifying ROS through antioxidant enzymes and ROS
scavenger molecules (Chen et al. 2005; Lin et al. 2016).
Although glial cells are equipped with high levels of protec-
tive and antioxidant compounds, astrocyte cell death has been
reported in human and animal models of brain injuries (Ishii
et al. 2017; Liu et al. 2016). As loss of glial cells may strongly
affect neuronal survival, protection of astrocytes from oxida-
tive insults appears essential to maintain brain functions.
The octadecaneuropeptide (ODN) is a peptide generated
through the proteolytic cleavage of the 86-amino acid precur-
sor diazepam-binding inhibitor (DBI) (Guidotti et al. 1983),
which is exclusively expressed in astroglial cells in the central
nervous system of mammals (Tonon et al. 2006; Tonon et al.
2013). DBI and its derived peptides, including ODN, belong
to the endozepine family. The primary structure of ODN has
been strongly preserved during evolution, suggesting that this
peptide exerts a wide range of biological functions. In fact,
ODN is involved in the control of food intake, sleep, aggres-
siveness, and anxiety (Tonon et al. 2013). At the cellular level,
numerous data indicate that ODN acts both as an autocrine
and paracrine factor modulating proliferation and differentia-
tion of astrocytes and neurons (Alfonso et al. 2012; Dumitru
et al. 2017). In addition, ODN exerts a potent protective effect
against the deleterious action of oxidative stress in cultured
astrocytes (Ghouili et al. 2018; Hamdi et al. 2011; Hamdi et al.
2012) and neurons (Kaddour et al. 2013). More recently, the
deficiency of the ODN precursor, in an in vivo model of PD,
was reported to increase sensitivity of dopaminergic neurons
in SNpc and ventral tegmental area (VTA) to MPTP neuro-
toxicity (Bahdoudi et al. 2018). Thus, ODN may be an endog-
enous peptide with neuroprotective and antioxidant properties
on astrocytes.
It is well established that 6-OHDA provokes cell apoptosis
through the production of ROS, leading to apoptosis and ac-
tivation of caspase-3 in both neurons and glial cells
(Hernandez-Baltazar et al. 2013; Mladenovic et al. 2004).
Since, the gliopeptide ODN protects granule neurons against
apoptosis induced by 6-OHDA exposure (Kaddour et al.
2013), we hypothesized that ODN may also protect astrocytes
J Mol Neurosci
against 6-OHDA oxidative insult. Therefore, the purpose of
the present study was to examine the potential protective ac-
tion of ODN against 6-OHDA-induced astrocyte cell death
and to investigate the effects of the peptide on some oxidative
stress-related parameters.
Materials and Methods
Animals
Wistar rats (Pasteur Institute, Tunis, Tunisia, and Charles
River Laboratories, St Germain sur l’Arbresle, France) were
kept in a temperature-controlled room (21 ± 1 °C) under an
established photoperiod (lights on from 7:00 am to 7:00 pm)
with free access to food and water. Experiments were per-
formed in accordance with the American Veterinary Medical
Association. Approval for these experiments was obtained
from the Medical Ethical Committee For the Care and Use
of Laboratory Animals of Pasteur Institute of Tunis
(Approval No. FST/LNFP/Pro152012).
Chemicals
Dulbecco’s modified Eagle’s medium (DMEM), D(+)-glu-
cose, F-12, L-glutamine, foetal bovine serum (FBS), N-2-
hydroxyethylpiperazine-N-2-ethane sulfonic acid buffer solu-
tion (HEPES), antibiotic-antimycotic solution, and trypsin-
EDTA were purchased from Gibco (Invitrogen, Grand Island
NY, USA). 6-OHDA, fluorescein diacetate-acetoxymethyl es-
ter (FDA-AM), bovine serum albumin (BSA),
nitrotetrazolium blue chloride (NBT), Triton X-100, bovine
liver catalase, DL-epinephrine, dimethylsulfoxyde (DMSO),
dihydrorhodamine 123 (DHR123), lactate dehydrogenase
(LDH) assay kit, and insulin were obtained from Sigma-
Aldrich (St. Louis, MO, USA). 5-6-chloromethyl-2′,7′-
dichlorodihydrofluorescein diacetate, acetyl ester (CM-
H2DCFDA), dihydroethidium (DHE), and 5,5″,6,6″-
tetrachloro-1,1″,3,3″-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1) were from Molecular Probes (Eugene, Oregon,
USA). The Apo-ONE homogeneous caspase-3/7 assay kit
was purchased from Promega (Charbonnières, France). Rat
ODN (QATVGDVNTDRPGLLDLK) was synthesized by
using the standard fluorenylmethyloxycarbonyl (Fmoc) pro-
cedure, as previously described (Leprince et al. 2001).
Secondary Culture of Cortical Rat Astrocytes
Secondary cultures of rat cortical astrocytes were prepared
from newborn Wistar rats of both sexes as previously de-
scribed (Douiri et al., 2016) with minor modifications.
Briefly, cerebral hemispheres were collected in DMEM/F12
(2:1; v/v) culture medium supplemented with 2 mM
glutamine, 1‰ insulin, 5 mM HEPES, 0.4% glucose, and
1% of the antibiotic-antimycotic solution. The tissues were
dissociated mechanically with a syringe equipped with a 1-
mm gauge needle, and filtered through a 100-μm sieve
(Falcon, Franklin Lakes, NJ, USA). Dissociated cells were
resuspended in culture medium supplemented with 10%
FBS, plated in 75-cm2 flasks (Greiner Bio-one GmbH,
Frickenhausen, Germany) and incubated at 37 °C in a 5%
CO2/95% air. When cultures were confluent, astrocytes were
isolated by shaking overnight the flasks with an orbital agita-
tor. Adhesive cells were detached by trypsination and
preplated for 5 min to discard contaminating microglial cells.
Then, the non-adherent astrocytes were harvested and plated
on 35-mm Petri dishes or 24-well plates at a density of 42,000
cells/cm2. After 5 days (DIV5), more than 98% of the cells
were labeled with antibodies against glial fibrillary acidic pro-
tein (Douiri et al. 2016). All experiments were performed on
5- to 7-day-old secondary cultures.
Cell Cytotoxicity Measurement
Cultured astrocytes seeded into 24-well plates were incubated
at 37 °C with fresh serum-free culture medium in the absence
or presence of test substances. At the end of the incubation,
membrane integrity was assessed as a function of the amount
of cytoplasmic LDH released into the medium with a LDH
assay kit, according to the manufacturer’s instructions. LDH
activity was measured at 340 nm with a spectrophotometric
microplate reader (Bio-Rad Laboratories, Philadelphia, USA).
The results were expressed as a percentage of total LDH re-
lease after cell lysis with 1% Triton X-100 in saline phosphate
buffer (PBS, 0.1 M, pH 7.4).
Cell Survival Measurement
Cultured astrocytes seeded into 24-well plates were incubated
at 37 °C with fresh serum-free culture medium in the absence
or presence of test substances. Cell survival was quantified by
measuring FDA in cultured astrocytes. At the end of the incu-
bation, cells were incubated in the dark with FDA-AM (15 μg/
mL, 8 min, 37 °C), rinsed twice with PBS (0.1 M, pH 7.4), and
lysed with a 10 mM Tris–HCl solution containing 1% sodium
dodecyl sulfate (SDS). Fluorescence intensity (λ excitation =
485 nm and λ emission = 538 nm) was measured with a
FL800TBI fluorescence microplate reader (Bio-Tek
Instruments, Winooski, VT, USA).
Measurement of Mitochondrial Activity
Cultured astrocytes seeded into 24-well plates were incubated
at 37 °C with fresh serum-free culture medium in the absence
or presence of test substances. Mitochondrial membrane po-
tential was quantified using the JC-1 probe. At the end of the

experiments, astrocytes were treated for 15 min with the JC-1
probe (10 μg/mL) and then washed twice with PBS.
Fluorescence intensity was measured with a FL800TBI fluo-
rescence microplate reader and expressed as a ratio of the
fluorescence emission at 590 nm (orange, intact mitochondrial
membrane potential) versus 530 nm (green, collapsed mito-
chondrial membrane potential).
Caspase-3 Activity Measurement
Cultured astrocytes seeded into 24-well plates were incubated
at 37 °C with fresh serum-free culture medium in the absence
or presence of test substances. At the end of the experiments,
cells were washed twice with PBS and caspase-3 activity was
measured with a caspase-3 assay system (Apo-ONE
Homogeneous Caspase-3/7 kit, Promega). Cells were resus-
pended in DMEM (100 μL) mixed with 1X caspase assay
buffer containing 25 μM caspase-3 substrate. Caspase-3 ac-
tivity was calculated from the slope of the fluorescence mea-
sured every 15 min for 3 h with excitation at 485 nm and
emission at 530 nm, and expressed as a percentage of the
control.
Intracellular Reactive Oxygen Species Measurement
ROS were detected by measuring the fluorescence of 2′,7′-
dichlorodihydrofluorescein (DCF) resulting from the
deacetylation and oxidation of the non-fluorescent compound
DCFH2-DA. Cells seeded into 24-well plates were exposed to
6-OHDA with or without ODN, incubated with 10 μM cell-
permeant DCFH2DA in serum-free loading medium for
30 min at 37 °C and then washed twice with PBS. DCF fluo-
rescence (λ excitation = 485 nm and λ emission = 538 nm)
was measured with a FL800TBI fluorescence microplate read-
er, and data were analyzed by Gen5 Data Analysis Software.
Intracellular Peroxide Measurement
H 2 O 2 and/or OH ° generation were detected by using
DHR123, which is a cell-permeable non-fluorescent com-
pound that is oxidized by cellular peroxides to fluorescent
rhodamine 123 (Rh 123) and exhibits green fluorescence.
Astrocytes were incubated at 37 °C with fresh serum-free
medium in the absence or presence of 6-OHDA with or with-
out ODN. At the end of the incubation, cultures were washed
twice with PBS, to prevent oxidation of DHR123 by perox-
ides in the medium, and incubated at 37 °C for 15 min in fresh
medium containing 6 μM DHR123, and then washed twice
with PBS. Rh 123 fluorescence (λ excitation = 505 nm and λ
emission = 529 nm) was measured with a FL800TBI micro-
plate reader, and data were analyzed by Gen5 Data Analysis
Software.
J Mol Neurosci
Intracellular Superoxide Measurement
Superoxide levels were detected by measuring the fluores-
cence of ethidium, which derived from the oxidation of the
non-fluorescent compound dihydroethidium (DHE).
Astrocytes were incubated at 37 °C with fresh serum-free
medium in the absence or presence of 6-OHDA with or with-
out ODN. At the end of the incubation, cells were incubated
for 20 min in fresh medium containing 2 μM DHE at 37 °C
and then washed twice with PBS. Ethidium fluorescence (λ
excitation = 520 nm and λ emission = 610 nm) was measured
with a FL800TBI microplate reader, and data were analyzed
by Gen5 Data Analysis Software.
Respiratory Burst Assay
The formation of the O2°−, produced by respiratory burst, was
detected by the reduction of nitroblue tetrazolium (NBT) to
dark-blue formazan deposits. Astrocytes were incubated at
37 °C with fresh serum-free medium in the absence or pres-
ence of 6-OHDA with or without ODN. At the end of the
incubation, cells were washed once with PBS at 37 °C, and
then incubated for 2 h in the dark with a reaction mixture
containing NBT (1 mg/mL) and BSA (1 mg/mL). To visualize
the formation of blue crystals, cells were examined and im-
ages were acquired with an eclipse E-600 microscope (Nikon,
Champigny-sur-Marne, France) equipped with a 3 CCD Sony
DXC950 camera interfaced with the Visiolab computerized
program (Biocom, Les Ulis, France).
Measurement of Antioxidant Enzyme Activities
Cultured cells seeded into 35-mm Petri dishes were incubated
at 37 °C for 72 h with fresh serum-free medium in the absence
or presence of test substances. At the end of the incubation,
cells were washed twice with PBS and total cellular proteins
were extracted by using the lysis buffer containing 50 mM
Tris–HCl (pH 8), 10 mM EDTA, 100 μM phenylmethyl-
sulfonylfluoride, and 1% Triton X-100. The homogenate
was centrifuged (16,000 g, 4 °C, 20 min), and the cellular
extract contained in the supernatant was stored at − 20°C until
enzyme activity determinations.
SOD activity was measured using a spectrophotometric
assay, which consists in measuring epinephrine autoxidation
induced by superoxide anion. Samples, prepared as described
above, were incubated for 3 min with a mixture containing
bovine catalase (0.4 U/μL), DL-epinephrine (5 mg/mL), and
Na2CO3/NaHCO3 buffer (62.5 mM, pH 10.2). The oxidation
of epinephrine was measured at 480 nm with a Bio-Rad spec-
trophotometric microplate reader.
Catalase activity was determined on the basis of the de-
crease of H2O2. Samples, prepared as described above, were
J Mol Neurosci

mixed with 30 mM H2O2 in PBS. The disappearance of H2O2 Results

was measured at 240 nm for 180 s at 30-s intervals.
Glioprotective Effect of ODN Against 6-OHDA-Induced
Quantitative PCR Analysis Astroglial Cell Death

Cultured cells seeded into 35-mm Petri dishes were incubated
at 37 °C with fresh serum-free medium. At the end of the
incubation, the culture medium was removed and cells
were washed twice with PBS (0.1 M, pH 7.4). Total
RNA was extracted by using Tri reagent (Sigma, St
Quentin Fallavier, France) and purified using the
NucleoSpin kit (Macherey-Nagel, Hoerd, France).
cDNA was synthetized from 3 to 4 μg of total RNA
with ImProm II Promega kit (Promega). Quantitative
RT-PCR was performed on cDNA in the presence of a
1× Fast SYBR Green universal PCR Master mix
(Applied Biosystems, Courtaboeuf, France) containing
dNTPs, MgCl2, SYBR green reporter dye, and
AmpliTaq Gold DNA polymerase, with forward and re-
verse primers (Table 1), using an ABI Prism 7500
Sequence Detection System (Applied Biosystems). The
relative amount of cDNA in each sample was calculated
using the comparative quantification cycle (Cq) method
and expressed as 2−ΔΔCq using GAPDH as an internal
control.
Statistical Analysis
Data are expressed as the mean ± SEM from three indepen-
dent experiments. Statistical analysis of the data was per-
formed by using ANOVA, followed by Bonferroni’s test. A
p value of 0.05 or less was considered as statistically
significant.
Administration of 6-OHDA on cultured astrocytes in-
duced a time- and dose-dependent cell death (Fig. 1a,
b). The concentration of 120 μM 6-OHDA, which killed
50% of the cells within 72 h of treatment, was used in
subsequent experiments. In these conditions, co-
incubation of cells with 6-OHDA and graded concentra-
tions of ODN (10−14 to 10−8 M) provoked mirror pro-
tective effects on astroglial survival and LDH leakage
(Fig. 1c). The glioprotection of ODN was concentration-
dependent in the range 10−11 to 10−8 M, and a complete
reversal of the toxic effect of 6-OHDA was obtained
with subnanomolar concentrations of ODN (Fig. 1c).
Incubation of astrocytes with ODN alone did not affect
cell survival whatever the time or the dose.
ODN Exerts its Glioprotective Effect
Through the Intrinsic Mitochondrial and Caspase-3
Pathway
As previously reported in different types of cells (Jordan et al.
2004; Perfeito et al. 2013), 6-OHDA inhibited the mitochondrial
electron transport chain, measured with the fluorescent
ratiometric probe JC-1 (Fig. 2a, b), and caused caspase-3 acti-
vation (Fig. 2c) in a dose-dependent manner in astrocytes, phe-
nomena which lead to typical apoptotic cell death. The present
data showed that addition of glioprotective doses of ODN to the
incubation medium (10−10 and 10−9 M) totally suppressed the
deleterious effect of 6-OHDA on mitochondrial membrane

Table 1 Sequences of the primers

used for real-time PCR Gene GenBank accession number Sequence
experiments
Mn-SOD NM_017051.2
Forward 5-TGGACAAACCTGAGCCCTAA-3
Reverse 5-GACCCAAAGTCACGCTTGATA-3
CAT NM_012520.2
Forward 5-ATCAGGGATGCCATGTTGTT-3
Reverse 5-GGGTCCTTCAGGTGAGTTTG-3
Gpx1 NM_030826.4
Forward 5-GACATCAGGAGAATGGCAAGA-3
Reverse 5-CACCTCGCACTTCTCAAACA-3
Srxn1 NM_001047858.3
Forward 5-CAACGTGCCAATCGCCGTGC-3
Reverse 5-GGGTCCTCCAGGATCGTGTCCA-3
GAPDH NM_017008.4
Forward 5-CAGCCTCGTCTCATAGACAAGATG-3
Reverse 5-CAATGTCCAACTTTGTCACAAGAGAAA-3
 J Mol Neurosci

a 10−9 M ODN totally blocked toxin-induced caspase-3 activation

125 **** 100 (Fig. 2d).
****

LDH release (% of total LDH)

Cell viability (% of control)

100 80
ODN Prevents the Effect of 6-OHDA on Intracellular
* ROS Accumulation
75 * 60
*
It is known that the toxic effect of 6-OHDA is linked to an
50 40
**** over-generation of ROS, such as H2O2, O2°- and OH° (Blum
et al. 2000; Elkon et al. 2004). To examine whether ODN
****
25 20 could block 6-OHDA-induced intracellular ROS accumula-
6-OHDA (120 µM) - + + + + -
tion, astrocytes were first labeled with the CMH2DCFDA
24h 48h 72h 96h
probe, which generates the fluorescent compound DCF upon
b ****
oxidation with ROS. Incubation of cells with graded concen-
100 **** 100 trations of 6-OHDA (10 to 200 μM) for 72 h induced a dose-
dependent increase in DCF fluorescence intensity (Fig. 3a).

LDH release (% of total LDH)

Cell viability (% of control)

**
80 80 For doses of 10−10 and 10−9 M, ODN did not modify DCF
***
fluorescence intensity, but totally abolished the effect of
60 60 120 μM 6-OHDA on DCF formation (Fig. 3b).
*
**** We also examined the effect of ODN on 6-OHDA-induced
40 ****
40 H2O2 formation in astrocytes by measuring DHR123 fluores-
cence intensity. Treatment of astroglial cells with toxic con-
20 20
centrations of 6-OHDA (60 to 200 μM) induced a dose-
dependent increase in DHR123 fluorescence intensity
10 20 30 60 120 200
6-OHDA [µM]
(Fig. 3c). Co-treatment of the cells with ODN (10−10 or
c 10−9 M) totally suppressed the effect of the toxin (10 μM to
100 100
120 μM) on peroxide production (Fig. 3c–d).
LDH release (% of total LDH)

####
Cell viability (% of control)

####
80
#
75 ##

ODN Prevents the Effect of 6-OHDA on Superoxide

50
25
-14 -13 -12
log M [ODN]
Fig. 1 Glioprotective effect of ODN on 6-OHDA-induced astrocyte cell
death. a Effect of 6-OHDA (120 μM) on astrocyte survival after 24, 48,
72, and 96 h of treatment. b Effect of graded concentrations of 6-OHDA
(10 to 200 μM) on astrocyte survival after 72 h. c Effect of graded
concentrations of ODN (10−14 to 10−8 M) on 6-OHDA (120 μM)-induced
astrocyte cell death after 72 h of treatment. Cell survival (___) was quan-
tified by measuring FDA fluorescence intensity, and the results are
expressed as percentages of the control (empty circle). Cell death (- - -)
was determined by measuring LDH activity in culture medium, and the
results are expressed as percentages of total LDH released from Triton-
lysed cells (gray triangle). Each value is the mean (± SEM) from at least
18 different wells from 3 independent cultures. ANOVA followed by
Bonferroni’s test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs.
control. #P < 0.05; ##P < 0.01; ####P < 0.0001 vs. 6-OHDA-treated cells
potential (Fig. 2b). To further explore the mechanism involved
in the protective action of ODN, we have monitored its effect on
caspase-3 activity. Co-treatment of the cells with 10−10 and
**
***
60 Radical Generation
***
****
40 Since large amounts of O2°− are produced by respiratory burst
under a drastic oxidative stress status, we have investigated
20 the effect of ODN on 6-OHDA-induced O2°− formation by
-11 -10 -9 -8
astroglial cells. Control astrocytes exhibited very few blue
precipitates (reflecting reduction of NBT by O2°- production)
in cell bodies (Fig. 4a). Treatment of astrocytes with 120 μM
6-OHDA resulted in a marked increase of the respiratory burst
as shown by the labeling of most cell bodies in blue (Fig. 4a).
In contrast, only few cells exhibited blue labeling after co-
administration of ODN (10−10 M) and 6-OHDA (120 μM)
(Fig. 4a). Quantitative analysis using the DHE probe indicated
that incubation of cultured astrocytes with graded concentra-
tions of 6-OHDA (10–200 μMM) for 72 h, induced a dose-
dependent increase in DHE fluorescence intensity (Fig. 4b).
Addition of ODN (10−10 M) to the incubation medium totally
blocked the effect of moderate concentrations of 6-OHDA
(from 30 to 120 μM) and partially reversed the effect of a
higher concentration of 6-OHDA (200 μM) on DHE forma-
tion (Fig. 4b). In addition, co-treatment of the cells with ODN
(10−10 or 10−9 M) restored the O2°− levels to the control values
in 120 μM 6-OHDA-treated cells (Fig. 4c).
J Mol Neurosci

Control Control
a 6-OHDA
6 OHDA b 6-OHDA
6 OHDA
6 OHDA + ODN
6-OHDA 6-OHDA + ODN
125 # ### ### ### 12
125

ontrrol)
ontrrol)

####
100 100 ####

( of co
atio (% of co

*
*

m ratio (%
75 *** 75
###
m ra

50 50

30 nm
30 nm

**** ***

0/53
0/53

25 **** 25

590
590

5
5

0 0
ODN (10-10
10M) - - + - + - + - + - + 6-OHDA
O ((120 µM)) - + + + + +
6-OHDA
6 OHDA 6
6-OHDA
OHDA 66-OHDA
OHDA 66-OHDA
OHDA 6 6-OHDA
OHDA -12 -10 -9 -8
(10 µM) (30 µM) (60 µM) (120 µM) (200 µM) log M [ODN]

Control
C t l Control
C t l
6-OHDA 6-OHDA
c 6-OHDA + ODN d 6-OHDA + ODN

# ### #### ###

250 ****
ontrrol)

250
ontrrol))

**** ****
3 activity (% of co
y (% of co

200 200
***
*
##

150 * 150
vity
3 activ

#### ####
100 100
se-3
pase-3

pas

50 50
asp
asp

Ca
Ca

0 0
ODN (10-10M) - - + - + - + - + - + 6-OHDA (120 µM) - + + + + +
6-OHDA 6-OHDA 6-OHDA 6-OHDA 6-OHDA -12 -10 -9 -8
(10 µM) (30 µM) (60 µM) (120 µM) (200 µM) log M [ODN]

Fig. 2 Effect of ODN on 6-OHDA-induced impairment of mitochondrial
transmembrane potential and activation of caspase-3 in astrocytes after
72 h of treatment. a, c Effect of graded concentrations of 6-OHDA (10 to
200 μM) on mitochondrial transmembrane potential and caspase-3 activ-
ity in astrocytes. b, d Effect of graded concentrations of ODN (10−12 to
10−8 M) on 6-OHDA (120 μM)-induced impairment of mitochondrial
transmembrane potential and activation of caspase-3 in astrocytes. Cells
mitochondrial transmembrane potential was assessed by using the JC-1
probe and the ratio of fluorescence emissions 590/530 nm was measured
as an index of mitochondrial activity. Caspase-3 activity was quantified
by measuring the fluorescence of the caspase substrate, Z-DEVD-
Rhodamine 110. The results are expressed as percentages of the control.
Each value is the mean (± SEM) of at least 12 different wells from 3
independent cultures. ANOVA followed by Bonferroni’s test: *P < 0.05;
***
P < 0.001; **** P < 0.0001 vs. control. # P < 0.05; ## P < 0.01;
###
P < 0.001; ####P < 0.0001 vs. 6-OHDA-treated cells
 J Mol Neurosci

a b Control
250 Control 250 6-OHDA
6 OHDA
****
6O
6-OHDA 6-OHDA + ODN

n
ation
on
ulattio 6-OHDA + ODN
#
200 **** 200 ****
ns

umula
#

ntrrol))
mu

* #
ol)
cum

**

ccu
onttro

150 * # 150 # #

con
#
acc

OS ac
ns # # # #
ns
% of co

o c
Sa

# # #

% of
C lullarr ROS

RO
100

ar R
100

(%
(%

ula
ellu
Ce
50
Cel

50

0 0
ODN (10-10M) - - + - + - + - + - + 6-OHDA - + + + + +

6
6-OHDA
OHDA 10 µM 30 µM 60 µM
M 120 µM
M 200 µM
M (120 µM) -12
12 -10
10 -9 9 -88
log M [ODN]

c d
Control Control
6 OHDA
6-OHDA 6 OHDA
6-OHDA
ntrrol))

2 0
250 250
*** ntrrol))
6-OHDA + ODN 6-OHDA + ODN
con

#
con
o c

200 # 200 ***

% of

o c

**** # #
****
% of
nsity (%

** # #
nsiity (%

#
# # #
150 # 150 #
# # #
#
3 intten

ns ns # # #
#
DHR 123 intten

100 100
R1
DHR 123

50 50
D

0 0
ODN (10-10 M) - - + - + - + - + - + 6 OHDA
6-OHDA - + + + + +
6-OHDA
6 OHDA M
10 µM 30 µM 60 µM
M 120 µM
M 200 µM
M (120 µM) 12 -10
-12 10 -99 -8
8
log M [ODN]
Fig. 3 Effect of ODN on 6-OHDA-induced intracellular generation of DCF and DHR 123 fluorescence, respectively. The results are expressed
ROS and accumulation of H2O2. Cells were incubated for 72 h with as percentages of control. Each value is the mean (± SEM) of at least 12
graded concentrations of 6-OHDA (10 to 200 μM) in the absence or different wells from 3 independent cultures. ANOVA followed by
presence of ODN (10−10 M; a, c). Cells were co-incubated with graded Bonferroni’s test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs.
concentrations of ODN (10−12 to 10−8 M) and 6-OHDA (120 μM; b, d). control. #P < 0.05; ##P < 0.01; ###P < 0.001; ns, not statistically different
Cellular ROS and H2O2 generation were quantified by measurement of vs. 6-OHDA-treated cells

ODN Blocks 6-OHDA-Induced Inhibition generated by 6-OHDA, we monitored the activities of

of Antioxidant Enzyme Activities in Cultured the two antioxidant enzymes SOD and catalase in astro-
Astrocytes cytes. Incubation of cultured cells with graded concen-
trations of 6-OHDA (10–200 μM; 72 h) induced a
To further explore the mechanism involved in the pro- dose-dependent decrease of both SOD (Fig. 5a) and
tective action of ODN against the oxidative stress catalase (Fig. 5b) activities. At higher concentrations
J Mol Neurosci

Control
a b
6-OHDA
250 6-OHDA
6 OHDA + ODN

c ntrrol)
Control
200 **** ****

o co
****
***

% of
#
# #

sitty ((%
150 ns # # #
# #

ens
100

nte
E in
DHE
50

DH
6-OHDA
6 OHDA (120 µM)
0
ODN (10-100M) - - + - + - + - + - +
6-OHDA 10 µM 30 µM 60 µM 120 µM 200 µM

c
250
ntrroll)

***
ns
200
con
% of c

#
6 OHDA+ODN (10-10 M)
6-OHDA+ODN #
#
150 #
y ((%

#
#
sity
ens

100
nte
HE in

50
DH
D

0
6-OHDA - + + + + +
(120 µM) -12
12 -10
10 -99 -8
8
log M [ODN]
Fig. 4 Effect of ODN on 6-OHDA-induced superoxide radical produc- 200 μM) in the presence or absence of ODN (10−10 M) (b). Cells were
tion. a Phase-contrast images illustrating the generation of superoxide incubated for 72 h with 120 μM 6-OHDA in the absence or presence of
radicals visualized with the presence of a dark-blue precipitate inside graded concentrations of ODN (10−12 to 10−8 M) (c). The results are
the cells. Cells were incubated for 72 h with medium alone (control) or expressed as percentages of control. Each value is the mean (± SEM) of
with 120 μM 6-OHDA alone or in the presence of ODN (10−10 M). Scale at least 12 different wells from 3 independent experiments. ANOVA
bar, 100 μm. b, c Cellular superoxide radical levels were quantified by followed by the Bonferroni’s test. ***P < 0.001; ****P < 0.0001 vs. con-
measurement of the fluorescence of oxidized dihydroethidium (DHE). trol. #P < 0.05; ###P < 0.001 vs. 6-OHDA-treated cells
Cells were incubated with increasing concentrations of 6-OHDA (10 to

of 6-OHDA tested, ODN (10−10 M) restored SOD and the inhibitory action of 120 μM 6-OHDA on the activity of
catalase activities to control values (Fig. 5a, b). endogenous antioxidant systems SOD (Fig. 5c) and catalase
Similarly, addition of graded concentrations of ODN (Fig. 5d).
(10−12–10−9 M) totally blocked in a dose-dependent manner
 J Mol Neurosci

a b
#
200 # 150 #

ntrol))
ntrroll) 6-OHDA ** 6-OHDA
#
#
#
*

on
6-OHDA + ODN 6-OHDA + ODN
*

% off co
con

150 125
( off c

y (%
y (%

vity
100 100
vity

a tiv
ctiv

e act
Daac

alase
50 * 75
SOD

**** **** **** *

Cata
S

Ca
0 50
10 20 30 60 120 200 10 20 30 60 120 200
6 OHDA [µM]
6-OHDA 6 OHDA [µM]
6-OHDA

Control Control
c 6-OHDA d 6 OHDA
6-OHDA
6 OHDA + ODN
6-OHDA 6-OHDA + ODN
#
# #
ol))

100 150 #
ol)

# #
ntro

#
onttro

# #
# #
on

#
% off co
o co

80 125
% of

#
# #
y (%
ctiivity (%

60 100
a tivity

***
**
e act

40 75
D ac

se
SOD

alas
SO

20 50
ata
ca

0 0
6-OHDA
6 OHDA - + + + + + + 6-OHDA
6 OHDA - + + + + + +
(120 µM) -12
12 -11
11 -10
10 -9
9 -8
8 (120 µM) -12
12 -11
11 -10
10 -9
9 -8
8
log
g M [[ODN]] log M [ODN]

Fig. 5 Effect of ODN on modulation of SOD and catalase activities in
cultured rat astrocytes. Cells were incubated with increasing
concentrations of 6-OHDA (10–200 μM) in the presence or absence of
ODN (10−10 M; a, b). Cells were incubated for 72 h with 120 μM of 6-
OHDA in the absence or presence of graded concentrations of ODN
(10−12 to 10−8 M; c, d). SOD activity was determined by measurement
of epinephrine autoxidation induced by superoxide anion, and catalase
activity by the decrease of H2O2. The results are expressed as percentages
of control. Each value is the mean (± SEM) calculated from at least 12
different dishes from 3 independent cultures. ANOVA followed by the
Bonferroni’s test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs.
control. #P < 0.05; ##P < 0.01; ###P < 0.001 different vs. 6-OHDA-treated
cells

ODN Inhibits 6-OHDA-Evoked Antioxidant Gene
Expression
Since 6-OHDA induces oxidative insults that facilitate
neurotoxicity, we examined the effect of ODN on the
expression of genes implicated in the control of oxida-
tive metabolism in 6-OHDA-treated astrocytes by means
of real-time PCR. Consistent with the literature (Khan
et al. 2010; Tuncel et al. 2012), the mRNA levels of
Mn-SOD (− 0.64%, ***P < 0.001), catalase (CAT; −
0.46%, **P < 0.01), and glutathione peroxidase (Gpx1,
−0.32%, *P < 0.05) were markedly decreased 6 h after
the beginning of the 6-OHDA treatment (Fig. 6a, c). As
illustrated in Fig. 6a, c, ODN alone, at a concentration
J Mol Neurosci

Control 6-OHDA Control 6-OHDA

a ODN 6-OHDA + ODN b ODN 6-OHDA + ODN
3 ###
3 *** ### ***
mRNA Mn-SOD levels ** ***

mRNA CAT levels

(% of control)
(% of control)
2 2

1 1
*** **

0 0
ODN (10-10M) - + - + ODN (10-10M) - + - +
6-OHDA (120 µM) 6-OHDA (120 µM)

c Control 6-OHDA d Control 6-OHDA

ODN 6-OHDA + ODN ODN 6-OHDA + ODN
###
4 4
*** ***
***
mRNA Srxn1 levels
###
mRNA GPx-1 levels

3 **
(% of control)
3
(% of control)

2 2

1 ** 1 **

0 0
ODN (10-10M) - + - + ODN (10-10M) - + - +

6-OHDA (120 µM) 6-OHDA (120 µM)

Fig. 6 Effect of ODN on 6-OHDA-induced inhibition of Mn-SOD, cat-
alase, glutathione peroxidase, and sulfiredoxin-1 mRNA levels in cul-
tured rat astrocytes. Cultured astrocytes were incubated for 6 h with
medium alone, 10−10 M ODN, 120 μM 6-OHDA, or 120 μM 6-OHDA
plus 10−10 M ODN. Mn-Superoxide dismutase (Mn-SOD; a), catalase
(CAT; b), glutathione peroxidase (Gpx1; c), and sulfiredoxin-1 (Srxn1
d) mRNA levels were measured by quantitative RT-PCR. Data were
corrected using the GAPDH signal as an internal control and the results
are expressed as a percentage of controls. Each value is the mean (± SEM)
of at least four different wells from three independent experiments.
ANOVA followed by the Bonferroni’s test. **P < 0.01; ***P < 0.001 vs.
control. ###P < 0.001 different vs. 6-OHDA-treated cells

of 10−10 M, provoked a significant increase in Mn-SOD
(+ 1.75%, ***P < 0.001), CAT (+ 1.33%, ***P < 0.001),
and Gpx-1 (+ 1.82%, ***P < 0.001) and restored Mn-
SOD, CAT, and Gpx1 mRNA levels above control
levels in 6-OHDA-treated astrocytes.
Since sulfiredoxin-1 (Srxn1) is a redox sensor that regulates
key cellular processes including antioxidant defense and cell
protection (Li et al. 2013; Wu et al. 2017), we examined the
effect of ODN on the expression of Srxn1 gene under oxidative
stress provoked by 6-OHDA. RT-qPCR experiments showed
that 6-OHDA (120 μM) induced a decrease of Srxn1 gene
transcription (− 0.32%, **P < 0.01). Interestingly, in ODN-
treated astrocytes a strong stimulation of Srxn1 gene transcrip-
tion was observed (+ 2.3%, ***P < 0.001). Furthermore, the
addition of ODN (10−10 M) to the culture media completely
restored Srxn1 mRNA expression above control levels in 6-
OHDA-treated astrocytes (Fig. 6d).
Discussion
It has already been reported that oxidative stress causes apo-
ptosis in various cell types, including neuronal cells (Amri
et al. 2017; Ishii et al. 2017; Tronel et al. 2013; Yang et al.
2017), and we have previously shown that ODN protects cer-
ebellar granule neurons against 6-OHDA-induced apoptosis
(Kaddour et al. 2013). Here, we demonstrate that ODN pro-
tects astroglial cells against 6-OHDA-induced cell death
through stimulation of endogenous antioxidant systems

leading to inhibition of ROS accumulation and caspase-3
activation.
6-OHDA, a neurotoxin classically used to mimic PD
in vivo and in vitro, is usually thought to cross the cell mem-
brane through dopamine uptake transporters but has also been
shown to induce apoptosis in various cell types that do not
express dopaminergic transporters, such as PC12 cells (Blum
et al. 2000). In agreement with this, we observed that exposure
of cultured astrocytes to 6-OHDA concentrations above
30 μM reduced astrocyte viability in a concentration, and
time-dependent manner. The resistance of astrocytes to lower
doses of the toxin can be ascribed to their lower sensitivity to
6-OHDA toxicity when compared to neurons (Blum et al.
2000; Blum et al. 2001; Kaddour et al. 2013). This can be
explained by the higher antioxidant capacity of astrocytes
(Baxter and Hardingham 2016; Ferrero-Gutierrez et al.
2008; Mullett et al. 2013).
The fact that 6-OHDA up to 120 μM did not provoke
cell death within the first 24 h supports the idea that at
least part of its astrocyte toxicity requires progressive ex-
tracellular auto-oxidation of 6-OHDA in the medium and
generation of free radicals (Hanrott et al. 2006).
Incubation of cultured astrocytes with subnanomolar con-
centrations of ODN dose-dependently protected cells
against 6-OHDA-induced injury, indicating that ODN is
a potent glioprotective agent. While the beneficial effect
of ODN against astrocyte cell death has already been re-
ported (Ghouili et al. 2018; Hamdi et al. 2011; Hamdi
et al. 2012; Hamdi et al. 2015), this is the first demon-
stration that ODN exerts a glioprotective action against 6-
OHDA-induced toxicity. Dose-response experiments
showed that ODN was effective at low concentrations
(in the nanomolar range) while, at higher doses, the pro-
tective action of the gliopeptide gradually declined. Such
a bell-shaped concentration-response curve has already
been observed with ODN on cell proliferation (Gandolfo
et al. 1999), neuron protection (Kaddour et al. 2013) and
intracellular calcium mobilization in cultured astrocytes
(Leprince et al. 2001). ODN acts through either a
central-type benzodiazepine receptor (CBR) associated
with the GABAA receptor complex (Ferrero et al. 1986)
or a metabotropic G protein-coupled receptor positively
coupled to phospholipase C (Patte et al. 1995) and
adenylyl cyclase (Hamdi et al. 2012). It has been reported
that the protective action of ODN against oxidative stress
on astrocytes and cerebellar granules neurons is blocked
by a selective ODN metabotropic receptor antagonist but
is not affected by a CBR antagonist (Hamdi et al. 2012;
Hamdi et al. 2015; Kaddour et al. 2013). Consistent with
this observation, the concentrations of ODN needed to
prevent the deleterious effects of 6-OHDA on astroglial
cells were similar to those which activate the metabotro-
pic receptor (Leprince et al. 2001). Altogether, these data
J Mol Neurosci
suggest that ODN inhibits 6-OHDA-induced astrocyte cell
death through activation of its metabotropic receptor, and
that attenuation of ODN action at high doses might in part
be due to desensitization its receptor.
It has previously been reported that extracellular auto-
oxidation of 6-OHDA produces a large array of highly reac-
tive species such as H2O2, O2°- and OH° which are likely
responsible for part of the toxicity of the molecule (Raicevic
et al. 2005a; Raicevic et al. 2005b). Nevertheless, it has also
been shown that astroglial cells express catecholamine trans-
porters (Huertas et al. 2015) which can promote cytosol accu-
mulation of 6-OHDA and H2O2 generation after deamination
of the toxin by mitochondrial monoamine oxidase.
Accordingly, intracellular accumulation of ROS i.e. H2O2
and O2°- was strongly increased in 6-OHDA-treated astro-
cytes, and administration of ODN in the culture medium at-
tenuated in a dose-dependent manner the pro-oxidant action of
6-OHDA. It is widely accepted that overproduction of ROS
can cause cell death by multiple mechanisms including dam-
age of mitochondria, leading to activation of mitochondria-
dependent apoptotic pathways (Bahdoudi et al. 2018;
Goswami et al. 2016; Masmoudi-Kouki et al. 2011). The pres-
ent study reveals that ODN blocked the increase of the respi-
ration rate and mitochondrial O2°− generation induced by 6-
OHDA. As a consequence, ODN prevented the effect of 6-
OHDA on the collapse of the mitochondrial potential and on
the stimulation of caspase-3 activity. These data are consistent
with previous studies showing that, in cultured astrocytes and
cerebellar neurons, ODN stimulates the expression of the anti-
apoptotic gene Bcl-2 and simultaneously blocks 6-OHDA or
H2O2-induced expression of the pro-apoptotic gene Bax to
promote cell survival (Hamdi et al. 2012; Kaddour et al.
2013). The fact that, in situ, 6-OHDA provokes the expression
of the active form of caspase-3 in astroglial cells in the SNpc
(Hernandez-Baltazar et al. 2013), together with the observa-
tion that 6-OHDA-induced upregulation of the Bax to Bcl-2
mRNA ratio, provide evidence for the implication of the in-
trinsic apoptotic pathway in the gliotoxic action of 6-OHDA.
ROS are another key player involved in the oxidative in-
sults produced by 6-OHDA, which can impede neuronal sur-
vival (Tong et al. 2018; Tuncel et al. 2012). For instance in
both the nigrostriatal region and cultured neuronal cells, 6-
OHDA treatment is associated with alteration of the redox
status with notably glutathione (GSH) levels depletion, in-
creased protein and lipid oxidation and decreased expression
and activities of antioxidant enzymes (Elkon et al. 2004;
Kaddour et al. 2013; Kuruvilla et al. 2013; Tong et al. 2018;
Tuncel et al. 2012), In agreement with these data, the present
study reveals that ODN totally suppressed the inhibitory effect
of 6-OHDA on SOD and catalase activities. Furthermore,
quantitative PCR analysis indicated that ODN enhanced genic
expression of endogenous antioxidant enzymes SOD2, cata-
lase, Gpx-1 and Srnx-1 and prevented the 6-OHDA-induced
J Mol Neurosci
decrease in expression of these genes involved in the control
of the oxidative stress. The fact that knocking down of DBI,
the ODN precursor, results in a decrease in antioxidant en-
zyme expression with an exacerbation of oxidative assaults
in cultured astrocyte (Ghouili et al. 2018) as well as increased
sensitivity to MPTP neurotoxicity in PD mouse model
(Bahdoudi et al. 2018), suggests that the gliopeptide ODN
could blunt intracellular ROS accumulation and dampen the
oxidative processes through an upregulation of ROS-
detoxifying enzymes. Overexpression of the transcription fac-
tor NF-E2-related factor (Nrf2), which binds to antioxidant
response element (ARE) (Jakel et al. 2007; Johnson et al.
2008) to induce antioxidant enzyme expression, has been
found to be protective against oxidative damage and to con-
tribute to protection of dopaminergic neurons from 6-OHDA-
induced toxicity in vivo and in vitro (Lev et al. 2013; Masaki
et al. 2017). Therefore, in vitro studies have shown that silenc-
ing Srxn1 results in decrease of NrF-2/ARE activity and in-
tracellular SOD and glutathione content (Zhou et al. 2015a) as
well as increased sensitivity to ischemia- and H2O2-induced
oxidative damage in astrocytes (Yu et al. 2015; Zhou et al.
2015a; Zhou et al. 2015b). Altogether, these observations in-
dicate that upregulation of Srxn1, which might be downstream
of Nrf2, may be involved in the stimulatory action of ODN on
antioxidant enzyme gene transcription as a way of defense
from oxidative assaults.
The importance of astroglial cells in the protection of neu-
rons against oxidative stress in diverse neuropathological con-
ditions such as stroke and Parkinson’s disease is well
established. Several studies have shown that astroglial-
derived peptides, i.e. activity-dependent neurotrophic factor,
activity-dependent neuroprotective protein and endozepine
ODN, provide neuroprotection against various neurotoxins
including 6-OHDA (Dejda et al. 2005; Kaddour et al. 2013;
Masmoudi-Kouki et al. 2007), MPTP (Bahdoudi et al. 2018)
and alcohol (Sari and Gozes 2006). Besides its neuroprotec-
tive action, ODN is able to stimulate neurogenesis in the adult
mouse brain, and silencing the ODN precursor, DBI, leads to
growth arrest and neuro-progenitor cell death (Alfonso et al.
2012; Dumitru et al. 2017), suggesting that ODN plays a
major role during brain development and on neuron-glia in-
teraction. In support of this notion, it has been shown that, in
the cerebellar cortex, the ODN precursor is exclusively
expressed by Bergmann glia (Tonon et al. 1990), and alter-
ation of these cells by 6-OHDA treatment reduces DBI ex-
pression and disturbs maturation and migration of granule
neuron precursors (Podkletnova et al. 2001). Moreover,
DBI−/− mice are more sensitive to MPTP-induced inflamma-
tion, ROS accumulation and oxidative brain injuries
(Bahdoudi et al. 2018). Although the mechanisms are not
established, these data suggest that, in vivo, ODN acts as an
autocrine and/or paracrine factor important for the protection
of both astrocytes and neurons. As astrocytes and neurons are
in close interaction in the brain, future studies should focus on
animal models and/or in vitro culture models where the effect
of ODN can be investigated in an integrated manner on both
cell types.
In conclusion, the present study has demonstrated that ex-
posure of cultured astrocytes to 6-OHDA reduces in a dose-
and time-dependent manner cell viability, induces a strong
increase of ROS production and inhibits both SOD and cata-
lase activities. ODN exerts its protective effect upon the dele-
terious action of oxidative stress by stimulation of expression
of endogenous antioxidant enzymes (Mn-SOD, CAT, Gpx1
and Srnx1) that prevent ROS accumulation, preserve mito-
chondrial function and inhibit caspase-3 activation.
Collectively, these data suggest that astroglia-secreted ODN
acts as an autocrine and/or paracrine protective agent that may
have therapeutic potential for the treatment of cerebral injuries
involving oxidative neurodegeneration.
Acknowledgments This work was supported by the France-Tunisia
CMCU-Campus France/PHC Utique 16G0820/34940PK exchange pro-
gram (to Olfa Masmoudi-Kouki and David Vaudry), Alternance scholar-
ship of Tunisian Higher Education Ministry and the Laboratory of
Neurophysiology, Cellular Physiopathology and Biomolecules
Valorisation. LR18ES03, INSERM (U1239), the European Regional
Development Fund (PACT-CBS and PHEDERCPG projects), and the
Region Normandy. Europe gets involved in Normandy with European
Regional Development Fund (ERDF). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of
the manuscript.
References
Afshin-Majd S, Bashiri K, Kiasalari Z, Baluchnejadmojarad T, Sedaghat
R, Roghani M (2017) Acetyl-l-carnitine protects dopaminergic
nigrostriatal pathway in 6-hydroxydopamine-induced model of
Parkinson's disease in the rat. Biomed Pharmacother 89:1–9.
https://doi.org/10.1016/j.biopha.2017.02.007
Alfonso J, Le Magueresse C, Zuccotti A, Khodosevich K, Monyer H
(2012) Diazepam binding inhibitor promotes progenitor prolifera-
tion in the postnatal SVZ by reducing GABA signaling. Cell Stem
Cell 10:76–87. https://doi.org/10.1016/j.stem.2011.11.011
Amri F, Ghouili I, Amri M, Carrier A, Masmoudi-Kouki O (2017)
Neuroglobin protects astroglial cells from hydrogen peroxide-
induced oxidative stress and apoptotic cell death. J Neurochem
140:151–169. https://doi.org/10.1111/jnc.13876
Bahdoudi S, Ghouili I, Hmiden M, do Rego JL, Lefranc B, Leprince J,
Chuquet J, do Rego JC, Marcher AB, Mandrup S, Vaudry H, Tonon
MC, Amri M, Masmoudi-Kouki O, Vaudry D (2018)
Neuroprotective effects of the gliopeptide ODN in an in vivo model
of Parkinson’s disease. Cell Mol Life Sci 75:2075–2091. https://doi.
org/10.1007/s00018-017-2727-2
Baxter PS, Hardingham GE (2016) Adaptive regulation of the brain’s
antioxidant defences by neurons and astrocytes. Free Radic Biol
Med 100:147–152. https://doi.org/10.1016/j.freeradbiomed.2016.
06.027
Blesa J, Przedborski S (2014) Parkinson’s disease: animal models and
dopaminergic cell vulnerability. Front Neuroanat 8:155. https://doi.
org/10.3389/fnana.2014.00155

Blum D, Torch S, Nissou MF, Benabid AL, Verna JM (2000)
Extracellular toxicity of 6-hydroxydopamine on PC12 cells.
Neurosci Lett 283:193–196
Blum D, Torch S, Nissou MF, Verna JM (2001) 6-hydroxydopamine-
induced nuclear factor-kappa B activation in PC12 cells. Biochem
Pharmacol 62:473–481
Burda JE, Bernstein AM, Sofroniew MV (2016) Astrocyte roles in trau-
matic brain injury. Exp Neurol 275(Pt 3):305–315. https://doi.org/
10.1016/j.expneurol.2015.03.020
Chen LW, Yung KL, Chan YS (2005) Reactive astrocytes as potential
manipulation targets in novel cell replacement therapy of
Parkinson’s disease. Curr Drug Targets 6:821–833
Dallerac G, Rouach N (2016) Astrocytes as new targets to improve cog-
nitive functions. Prog Neurobiol 144:48–67. https://doi.org/10.
1016/j.pneurobio.2016.01.003
Dejda A, Sokolowska P, Nowak JZ (2005) Neuroprotective potential of
three neuropeptides PACAP, VIP and PHI. Pharmacol Rep 57:307–
320
Douiri S, Bahdoudi S, Hamdi Y, Cubi R, Basille M, Fournier A, Vaudry
H, Tonon MC, Amri M, Vaudry D, Masmoudi-Kouki O (2016)
Involvement of endogenous antioxidant systems in the protective
activity of pituitary adenylate cyclase-activating polypeptide against
hydrogen peroxide-induced oxidative damages in cultured rat astro-
cytes. J Neurochem 137:913–930. https://doi.org/10.1111/jnc.13614
Dumitru I, Neitz A, Alfonso J, Monyer H (2017) Diazepam binding
inhibitor promotes stem cell expansion controlling environment-
dependent neurogenesis. Neuron 94(125–137):e125. https://doi.
org/10.1016/j.neuron.2017.03.003
Dzyubenko E, Gottschling C, Faissner A (2016) Neuron-glia interactions
in neural plasticity: contributions of neural extracellular matrix and
perineuronal nets. Neural Plast 2016:5214961. https://doi.org/10.
1155/2016/5214961
Elkon H, Melamed E, Offen D (2004) Oxidative stress, induced by 6-
hydroxydopamine, reduces proteasome activities in PC12 cells: im-
plications for the pathogenesis of Parkinson's disease. J Mol
Neurosci 24:387–400. https://doi.org/10.1385/JMN:24:3:387
Espinosa-Oliva AM, de Pablos RM, Sarmiento M, Villaran RF, Carrillo-
Jimenez A, Santiago M, Venero JL, Herrera AJ, Cano J, Machado A
(2014) Role of dopamine in the recruitment of immune cells to the
nigro-striatal dopaminergic structures. Neurotoxicology 41:89–101.
https://doi.org/10.1016/j.neuro.2014.01.006
Ferrero-Gutierrez A, Perez-Gomez A, Novelli A, Fernandez-Sanchez MT
(2008) Inhibition of protein phosphatases impairs the ability of as-
trocytes to detoxify hydrogen peroxide. Free Radic Biol Med 44:
1806–1816. https://doi.org/10.1016/j.freeradbiomed.2008.01.029
Ferrero P, Santi MR, Conti-Tronconi B, Costa E, Guidotti A (1986) Study
of an octadecaneuropeptide derived from diazepam binding inhibi-
tor (DBI): biological activity and presence in rat brain. Proc Natl
Acad Sci U S A 83:827–831
Gandolfo P, Patte C, Thoumas JL, Leprince J, Vaudry H, Tonon MC
(1999) The endozepine ODN stimulates [3H]thymidine incorpora-
tion in cultured rat astrocytes. Neuropharmacology 38:725–732
Gardaneh M, Gholami M, Maghsoudi N (2011) Synergy between gluta-
thione peroxidase-1 and astrocytic growth factors suppresses free
radical generation and protects dopaminergic neurons against 6-
hydroxydopamine. Rejuvenation Res 14:195–204. https://doi.org/
10.1089/rej.2010.1080
Garzon D, Cabezas R, Vega N, Avila-Rodriguez M, Gonzalez J, Gomez
RM, Echeverria V, Aliev G, Barreto GE (2016) Novel approaches in
astrocyte protection: from experimental methods to computational
approaches. J Mol Neurosci 58:483–492. https://doi.org/10.1007/
s12031-016-0719-6
Ghouili I, Bahdoudi S, Morin F, Amri F, Hamdi Y, Coly PM, Walet-
Balieu ML, Leprince J, Zekri S, Vaudry H, Vaudry D, Castel H,
Amri M, Tonon MC, Masmoudi-Kouki O (2018) Endogenous ex-
pression of ODN-related peptides in astrocytes contributes to cell
J Mol Neurosci
protection against oxidative stress: astrocyte-neuron crosstalk rele-
vance for neuronal survival. Mol Neurobiol 55:4596–4611. https://
doi.org/10.1007/s12035-017-0630-3
Gomide VC, Silveira GA, Chadi G (2005) Transient and widespread
astroglial activation in the brain after a striatal 6-OHDA-induced
partial lesion of the nigrostriatal system. Int J Neurosci 115:99–117
Goswami P, Gupta S, Biswas J, Sharma S, Singh S (2016) Endoplasmic
reticulum stress instigates the rotenone induced oxidative apoptotic
neuronal death: a study in rat brain. Mol Neurobiol 53:5384–5400.
https://doi.org/10.1007/s12035-015-9463-0
Gu XL, Long CX, Sun L, Xie C, Lin X, Cai H (2010) Astrocytic expres-
sion of Parkinson's disease-related A53T alpha-synuclein causes
neurodegeneration in mice. Mol Brain 3:12. https://doi.org/10.
1186/1756-6606-3-12
Guidotti A, Forchetti CM, Corda MG, Konkel D, Bennett CD, Costa E
(1983) Isolation, characterization, and purification to homogeneity
of an endogenous polypeptide with agonistic action on benzodiaze-
pine receptors. Proc Natl Acad Sci U S A 80:3531–3535
Hamdi Y, Kaddour H, Vaudry D, Bahdoudi S, Douiri S, Leprince J,
Castel H, Vaudry H, Tonon MC, Amri M, Masmoudi-Kouki O
(2012) The octadecaneuropeptide ODN protects astrocytes against
hydrogen peroxide-induced apoptosis via a PKA/MAPK-dependent
mechanism. PLoS One 7:e42498. https://doi.org/10.1371/journal.
pone.0042498
Hamdi Y, Kaddour H, Vaudry D, Leprince J, Zarrouk A, Hammami M,
Vaudry H, Tonon MC, Amri M, Masmoudi-Kouki O (2015)
Octadecaneuropeptide ODN prevents hydrogen peroxide-induced
oxidative damage of biomolecules in cultured rat astrocytes.
Peptides 71:56–65. https://doi.org/10.1016/j.peptides.2015.06.010
Hamdi Y, Masmoudi-Kouki O, Kaddour H, Belhadj F, Gandolfo P,
Vaudry D, Mokni M, Leprince J, Hachem R, Vaudry H, Tonon
MC, Amri M (2011) Protective effect of the octadecaneuropeptide
on hydrogen peroxide-induced oxidative stress and cell death in
cultured rat astrocytes. J Neurochem 118:416–428. https://doi.org/
10.1111/j.1471-4159.2011.07315.x
Han X, Zhu S, Wang B, Chen L, Li R, Yao W, Qu Z (2014) Antioxidant
action of 7,8-dihydroxyflavone protects PC12 cells against 6-
hydroxydopamine-induced cytotoxicity. Neurochem Int 64:18–23.
https://doi.org/10.1016/j.neuint.2013.10.018
Hanrott K, Gudmunsen L, O'Neill MJ, Wonnacott S (2006) 6-
hydroxydopamine-induced apoptosis is mediated via extracellular
auto-oxidation and caspase 3-dependent activation of protein kinase
Cdelta. J Biol Chem 281:5373–5382. https://doi.org/10.1074/jbc.
M511560200
Hernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D (2013)
Activation of GSK-3beta and caspase-3 occurs in nigral dopamine
neurons during the development of apoptosis activated by a striatal
injection of 6-hydroxydopamine. PLoS One 8:e70951. https://doi.
org/10.1371/journal.pone.0070951
Huertas A, Wessinger WD, Kucheryavykh YV, Sanabria P, Eaton MJ,
Skatchkov SN, Rojas LV, Maldonado-Martinez G, Inyushin MY
(2015) Quinine enhances the behavioral stimulant effect of cocaine
in mice. Pharmacol Biochem Behav 129:26–33. https://doi.org/10.
1016/j.pbb.2014.11.021
Ishii T, Takanashi Y, Sugita K, Miyazawa M, Yanagihara R, Yasuda K,
Onouchi H, Kawabe N, Nakata M, Yamamoto Y, Hartman PS, Ishii
N (2017) Endogenous reactive oxygen species cause astrocyte de-
fects and neuronal dysfunctions in the hippocampus: a new model
for aging brain. Aging Cell 16:39–51. https://doi.org/10.1111/acel.
12523
Jakel RJ, Townsend JA, Kraft AD, Johnson JA (2007) Nrf2-mediated
protection against 6-hydroxydopamine. Brain Res 1144:192–201.
https://doi.org/10.1016/j.brainres.2007.01.131
Johnson JA, Johnson DA, Kraft AD, Calkins MJ, Jakel RJ, Vargas MR,
Chen PC (2008) The Nrf2-ARE pathway: an indicator and
J Mol Neurosci
modulator of oxidative stress in neurodegeneration. Ann N Y Acad
Sci 1147:61–69. https://doi.org/10.1196/annals.1427.036
Jordan J, Galindo MF, Tornero D, Gonzalez-Garcia C, Cena V (2004)
Bcl-x L blocks mitochondrial multiple conductance channel activa-
tion and inhibits 6-OHDA-induced death in SH-SY5Y cells. J
Neurochem 89:124–133. https://doi.org/10.1046/j.1471-4159.
2003.02299.x
Kaddour H, Hamdi Y, Vaudry D, Basille M, Desrues L, Leprince J, Castel
H, Vaudry H, Tonon MC, Amri M, Masmoudi-Kouki O (2013) The
octadecaneuropeptide ODN prevents 6-hydroxydopamine-induced
apoptosis of cerebellar granule neurons through a PKC-MAPK-
dependent pathway. J Neurochem 125:620–633. https://doi.org/10.
1111/jnc.12140
Khan MM, Ahmad A, Ishrat T, Khan MB, Hoda MN, Khuwaja G, Raza
SS, Khan A, Javed H, Vaibhav K, Islam F (2010) Resveratrol atten-
uates 6-hydroxydopamine-induced oxidative damage and dopamine
depletion in rat model of Parkinson’s disease. Brain Res 1328:139–
151. https://doi.org/10.1016/j.brainres.2010.02.031
Kiasalari Z, Khalili M, Baluchnejadmojarad T, Roghani M (2016)
Protective effect of oral hesperetin againstunilateral striatal 6-
hydroxydopamine damage in the rat. Neurochem Res 41:1065–
1072. https://doi.org/10.1007/s11064-015-1796-6
Kuruvilla KP, Nandhu MS, Paul J, Paulose CS (2013) Oxidative stress
mediated neuronal damage in the corpus striatum of 6-
hydroxydopamine lesioned Parkinson’s rats: neuroprotection by se-
rotonin, GABA and bone marrow cells supplementation. J Neurol
Sci 331:31–37. https://doi.org/10.1016/j.jns.2013.04.020
L' Episcopo F, Tirolo C, Testa N, Caniglia S, Morale MC, Marchetti B
(2010) Glia as a turning point in the therapeutic strategy of
Parkinson’s disease. CNS Neurol Disord Drug Targets 9:349–372
L'Episcopo F, Tirolo C, Testa N, Caniglia S, Morale MC, Cossetti C,
D'Adamo P, Zardini E, Andreoni L, Ihekwaba AE, Serra PA,
Franciotta D, Martino G, Pluchino S, Marchetti B (2011) Reactive
astrocytes and Wnt/beta-catenin signaling link nigrostriatal injury to
repair in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of
Parkinson’s disease. Neurobiol Dis 41:508–527. https://doi.org/10.
1016/j.nbd.2010.10.023
Leprince J, Oulyadi H, Vaudry D, Masmoudi O, Gandolfo P, Patte C,
Costentin J, Fauchere JL, Davoust D, Vaudry H, Tonon MC (2001)
Synthesis, conformational analysis and biological activity of cyclic
analogs of the octadecaneuropeptide ODN: design of a potent
endozepine antagonist. Eur J Biochem 268:6045–6057
Lev N, Barhum Y, Ben-Zur T, Melamed E, Steiner I, Offen D (2013)
Knocking out DJ-1 attenuates astrocytes neuroprotection against 6-
hydroxydopamine toxicity. J Mol Neurosci 50:542–550. https://doi.
org/10.1007/s12031-013-9984-9
Li Q, Yu S, Wu J, Zou Y, Zhao Y (2013) Sulfiredoxin-1 protects PC12
cells against oxidative stress induced by hydrogen peroxide. J
Neurosci Res 91:861–870. https://doi.org/10.1002/jnr.23218
Lin CH, Wang CH, Hsu SL, Liao LY, Lin TA, Hsueh CM (2016)
Molecular mechanisms responsible for neuron-derived conditioned
medium (NCM)-mediated protection of ischemic brain. PLoS One
11:e0146692. https://doi.org/10.1371/journal.pone.0146692
Liu L, Liu L, Shi J, Tan M, Xiong J, Li X, Hu Q, Yi Z, Mao D (2016)
MicroRNA-34b mediates hippocampal astrocyte apoptosis in a rat
model of recurrent seizures. BMC Neurosci 17:56. https://doi.org/
10.1186/s12868-016-0291-6
Liu Z, Chopp M (2016) Astrocytes, therapeutic targets for neuroprotec-
tion and neurorestoration in ischemic stroke. Prog Neurobiol 144:
103–120. https://doi.org/10.1016/j.pneurobio.2015.09.008
Masaki Y, Izumi Y, Matsumura A, Akaike A, Kume T (2017) Protective
effect of Nrf2-ARE activator isolated from green perilla leaves on
dopaminergic neuronal loss in a Parkinson’s disease model. Eur J
Pharmacol 798:26–34. https://doi.org/10.1016/j.ejphar.2017.02.005
Masmoudi-Kouki O, Douiri S, Hamdi Y, Kaddour H, Bahdoudi S,
Vaudry D, Basille M, Leprince J, Fournier A, Vaudry H, Tonon
MC, Amri M (2011) Pituitary adenylate cyclase-activating polypep-
tide protects astroglial cells against oxidative stress-induced apopto-
sis. J Neurochem 117:403–411. https://doi.org/10.1111/j.1471-
4159.2011.07185.x
Masmoudi-Kouki O, Gandolfo P, Castel H, Leprince J, Fournier A, Dejda
A, Vaudry H, Tonon MC (2007) Role of PACAP and VIP in
astroglial functions. Peptides 28:1753–1760. https://doi.org/10.
1016/j.peptides.2007.05.015
Michel PP, Toulorge D, Guerreiro S, Hirsch EC (2013) Specific needs of
dopamine neurons for stimulation in order to survive: implication
for Parkinson disease. FASEB J 27:3414–3423. https://doi.org/10.
1096/fj.12-220418
Mirza B, Hadberg H, Thomsen P, Moos T (2000) The absence of reactive
astrocytosis is indicative of a unique inflammatory process in
Parkinson’s disease. Neuroscience 95:425–432
Mladenovic A, Perovic M, Raicevic N, Kanazir S, Rakic L, Ruzdijic S
(2004) 6-Hydroxydopamine increases the level of TNFalpha and
bax mRNA in the striatum and induces apoptosis of dopaminergic
neurons in hemiparkinsonian rats. Brain Res 996:237–245
Mullett SJ, Di Maio R, Greenamyre JT, Hinkle DA (2013) DJ-1 expres-
sion modulates astrocyte-mediated protection against neuronal oxi-
dative stress. J Mol Neurosci 49:507–511. https://doi.org/10.1007/
s12031-012-9904-4
Murakami S, Miyazaki I, Asanuma M (2018) Neuroprotective effect of
fermented papaya preparation by activation of Nrf2 pathway in as-
trocytes. Nutr Neurosci 21:176–184. https://doi.org/10.1080/
1028415X.2016.1253171
Osborn LM, Kamphuis W, Wadman WJ, Hol EM (2016) Astrogliosis: an
integral player in the pathogenesis of Alzheimer's disease. Prog
Neurobiol 144:121–141. https://doi.org/10.1016/j.pneurobio.2016.
01.001
Patte C, Vaudry H, Desrues L, Gandolfo P, Strijdveen I, Lamacz M,
To n o n M C ( 1 9 9 5 ) T h e e n d o z e p i n e O D N s t i m u l a t e s
polyphosphoinositide metabolism in rat astrocytes. FEBS Lett
362:106–110
Pekny M, Pekna M, Messing A, Steinhauser C, Lee JM, Parpura V, Hol
EM, Sofroniew MV, Verkhratsky A (2016) Astrocytes: a central
element in neurological diseases. Acta Neuropathol 131:323–345.
https://doi.org/10.1007/s00401-015-1513-1
Perfeito R, Cunha-Oliveira T, Rego AC (2013) Reprint of: revisiting
oxidative stress and mitochondrial dysfunction in the pathogenesis
of Parkinson disease-resemblance to the effect of amphetamine
drugs of abuse. Free Radic Biol Med 62:186–201. https://doi.org/
10.1016/j.freeradbiomed.2013.05.042
Podkletnova I, Rothstein JD, Helen P, Alho H (2001) Microglial response
to the neurotoxicity of 6-hydroxydopamine in neonatal rat cerebel-
lum. Int J Dev Neurosci 19:47–52
Raicevic N, Mladenovic A, Perovic M, Harhaji L, Miljkovic D, Trajkovic
V (2005a) Iron protects astrocytes from 6-hydroxydopamine toxic-
ity. Neuropharmacology 48:720–731. https://doi.org/10.1016/j.
neuropharm.2004.12.003
Raicevic N, Mladenovic A, Perovic M, Miljkovic D, Trajkovic V (2005b)
The mechanisms of 6-hydroxydopamine-induced astrocyte death.
Ann N Y Acad Sci 1048:400–405. https://doi.org/10.1196/annals.
1342.049
Sandhu JK, Gardaneh M, Iwasiow R, Lanthier P, Gangaraju S, Ribecco-
Lutkiewicz M, Tremblay R, Kiuchi K, Sikorska M (2009)
Astrocyte-secreted GDNF and glutathione antioxidant system pro-
tect neurons against 6OHDA cytotoxicity. Neurobiol Dis 33:405–
414. https://doi.org/10.1016/j.nbd.2008.11.016
Sari Y, Gozes I (2006) Brain deficits associated with fetal alcohol expo-
sure may be protected, in part, by peptides derived from activity-
dependent neurotrophic factor and activity-dependent neuroprotec-
tive protein. Brain Res Rev 52:107–118. https://doi.org/10.1016/j.
brainresrev.2006.01.004

Soto-Otero R, Mendez-Alvarez E, Hermida-Ameijeiras A, Munoz-Patino
AM, Labandeira-Garcia JL (2000) Autoxidation and neurotoxicity
of 6-hydroxydopamine in the presence of some antioxidants: poten-
tial implication in relation to the pathogenesis of Parkinson’s dis-
ease. J Neurochem 74:1605–1612
Tong H, Zhang X, Meng X, Lu L, Mai D, Qu S (2018) Simvastatin
inhibits activation of NADPH oxidase/p38 MAPK pathway and
enhances expression of antioxidant protein in Parkinson disease
models. Front Mol Neurosci 11:165. https://doi.org/10.3389/
fnmol.2018.00165
Tonon MC, Desy L, Nicolas P, Vaudry H, Pelletier G (1990)
Immunocytochemical localization of the endogenous benzodiaze-
pine ligand octadecaneuropeptide (ODN) in the rat brain.
Neuropeptides 15:17–24
Tonon MC, Leprince J, Gandolfo P, Compère V, Pelletier G, Malagon
MM, Vaudry H (2006) Endozepines.In handbook of biologically
active peptides,. ed A J Kastin (New York: Elsevier):813–819
Tonon MC, Leprince J, Morin F, Gandolfo P, Compère V, Pelletier G,
Malagon M, Vaudry H (2013) Endozepines. In: Kastin AJ (ed)
Handbook of biological active peptides, 2nd edn. Elsevier, Oxford,
UK, pp 760–765
Toulorge D, Schapira AH, Hajj R (2016) Molecular changes in the post-
mortem parkinsonian brain. J Neurochem 139 Suppl 1:27–58.
https://doi.org/10.1111/jnc.13696
Tronel C, Rochefort GY, Arlicot N, Bodard S, Chalon S, Antier D (2013)
Oxidative stress is related to the deleterious effects of heme
oxygenase-1 in an in vivo neuroinflammatory rat model. Oxidative
Med Cell Longev 2013:264935. https://doi.org/10.1155/2013/
264935
Tuncel N, Korkmaz OT, Tekin N, Sener E, Akyuz F, Inal M (2012)
Antioxidant and anti-apoptotic activity of vasoactive intestinal
J Mol Neurosci
peptide (VIP) against 6-hydroxy dopamine toxicity in the rat corpus
striatum. J Mol Neurosci 46:51–57. https://doi.org/10.1007/s12031-
011-9618-z
Wachter B, Schurger S, Rolinger J, von Ameln-Mayerhofer A, Berg D,
Wagner HJ, Kueppers E (2010) Effect of 6-hydroxydopamine (6-
OHDA) on proliferation of glial cells in the rat cortex and striatum:
evidence for de-differentiation of resident astrocytes. Cell Tissue
Res 342:147–160. https://doi.org/10.1007/s00441-010-1061-x
Wu J, Chen Y, Yu S, Li L, Zhao X, Li Q, Zhao J, Zhao Y (2017)
Neuroprotective effects of sulfiredoxin-1 during cerebral ischemia/
reperfusion oxidative stress injury in rats. Brain Res Bull 132:99–
108. https://doi.org/10.1016/j.brainresbull.2017.05.012
Yang CM, Lin CC, Hsieh HL (2017) High-glucose-derived oxidative
stress-dependent heme oxygenase-1 expression from astrocytes con-
tributes to the neuronal apoptosis. Mol Neurobiol 54:470–483.
https://doi.org/10.1007/s12035-015-9666-4
Yu S, Wang X, Lei S, Chen X, Liu Y, Zhou Y, Zhou Y, Wu J, Zhao Y
(2015) Sulfiredoxin-1 protects primary cultured astrocytes from
ischemia-induced damage. Neurochem Int 82:19–27. https://doi.
org/10.1016/j.neuint.2015.01.005
Zhou Y, Duan S, Zhou Y, Yu S, Wu J, Wu X, Zhao J, Zhao Y (2015a)
Sulfiredoxin-1 attenuates oxidative stress via Nrf2/ARE pathway
and 2-Cys Prdxs after oxygen-glucose deprivation in astrocytes. J
Mol Neurosci 55:941–950. https://doi.org/10.1007/s12031-014-
0449-6
Zhou Y, Zhou Y, Yu S, Wu J, Chen Y, Zhao Y (2015b) Sulfiredoxin-1
exerts anti-apoptotic and neuroprotective effects against oxidative
stress-induced injury in rat cortical astrocytes following exposure
to oxygen-glucose deprivation and hydrogen peroxide. Int J Mol
Med 36:43–52. https://doi.org/10.3892/ijmm.2015.2205