Molecular mechanism underlying the cerebral effect of Gly-Pro-Glu tripeptide bound to L-dopa in a Parkinson's animal model

Article abstract of DOI:10.1007/s00726-011-1210-x

Oxidative stress is a critical contributing factor to neurodegenerative disorders. Therefore, the inhibition of ROS formation, responsible for chronic detrimental neuroinflammation, is an important strategy for preventing the neurodegenerative disease and for neuroprotective therapy. Gly-Pro-Glu (GPE) is the N-terminal tripeptide of insulinlike growth factor-I, which is naturally cleaved in the plasma and brain tissues. GPE has neuroprotective effects since it crosses the blood-CSF and the functional CSF- brain barriers and binds to glial cells. It has been shown that GPE improves motor behaviour in rats after 6-OHDA lesion, although it does not rescue dopaminergic neurons. Thus, we hypothesized that the GPE therapeutic efficacy in a Parkinson model might be improved by combining GPE to L-dopa. Here, we used an animal model that represents a progressive chronic Parkinson's disease (PD) model, characterized by high levels of oxidative stress and inflammation. We showed that the co-drug, in which L-dopa is covalently linked to the GPE tripeptide, by downregulating the expression of inflammatory genes, decreases the 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)- induced inflammatory response and, by up-regulating tyrosine hydroxylase, reduces MPTP-induced neurotoxicity. Furthermore, by determining the nuclear translocation/ activation of Nrf2 and NF-κB, we showed that systemic administration of the co-drug activates Nrf2- induced antioxidant response while suppressing NF-κB inflammatory pathway. Data suggest that the binding of L-dopa to GPE tripeptide might represent a promising strategy to supply L-dopa to parkinsonian patients.

Full text of DOI:10.1007/s00726-011-1210-x

Amino Acids (2012) 43:1359–1367
DOI 10.1007/s00726-011-1210-x
ORIGINAL ARTICLE
Molecular mechanism underlying the cerebral effect
of Gly-Pro-Glu tripeptide bound to L-dopa in a Parkinson’s
animal model
•
Alba Minelli C. Conte I. Cacciatore
•
•
•
C. Cornacchia F. Pinnen
Received: 8 September 2011 / Accepted: 22 December 2011 / Published online: 5 January 2012
Ó Springer-Verlag 2012
Abstract Oxidative stress is a critical contributing factor
to neurodegenerative disorders. Therefore, the inhibition of
ROS formation, responsible for chronic detrimental neur-
oinflammation, is an important strategy for preventing the
neurodegenerative disease and for neuroprotective therapy.
Gly-Pro-Glu (GPE) is the N-terminal tripeptide of insulin-
like growth factor-I, which is naturally cleaved in the
plasma and brain tissues. GPE has neuroprotective effects
since it crosses the blood–CSF and the functional CSF–
brain barriers and binds to glial cells. It has been shown
that GPE improves motor behaviour in rats after 6-OHDA
lesion, although it does not rescue dopaminergic neurons.
Thus, we hypothesized that the GPE therapeutic efficacy in
a Parkinson model might be improved by combining GPE
to L-dopa. Here, we used an animal model that represents a
progressive chronic Parkinson’s disease (PD) model,
characterized by high levels of oxidative stress and
inflammation. We showed that the co-drug, in which
L-dopa is covalently linked to the GPE tripeptide, by down-
regulating the expression of inflammatory genes, decreases
the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-
induced inflammatory response and, by up-regulating
tyrosine hydroxylase, reduces MPTP-induced neurotox-
icity. Furthermore, by determining the nuclear transloca-
tion/activation of Nrf2 and NF-jB, we showed that
systemic administration of the co-drug activates Nrf2-
induced antioxidant response while suppressing NF-jB
inflammatory pathway. Data suggest that the binding of
L-dopa to GPE tripeptide might represent a promising
strategy to supply L-dopa to parkinsonian patients.
Keywords MPTP ꢀ PD animal model ꢀ LD-GPE ꢀ
Nrf2 ꢀ NF-jB
Introduction
Gly-Pro-Glu (GPE) is the N-terminal tripeptide of insulin-
like growth factor-I, which is naturally cleaved in the
plasma and brain tissues (Sara et al. 1989; Batchelor et al.
2003; Baker et al. 2005; Sizonenko et al. 2001). It has long
been known that GPE has neuroprotective effects and, that,
following peripheral administration, crosses the blood–
CSF and the functional CSF–brain barriers and binds to
glial cells (Saura et al. 1999; Alexi et al. 1999; Guan et al.
1999, 2000, 2004; Sizonenko et al. 2001; Aguado-Llera
et al. 2004; Baker et al. 2005; Zhao et al. 2005; Shapira
et al. 2009). The effectiveness of central uptake of GPE
was confirmed by the improvement of functional deficits
after 6-OHDA lesion in rats (Krishnamurthi et al. 2004),
by the neuroprotective effects in the CA1-2 sub-regions
of the hippocampus of hypoxic–ischaemic injured rats
(Shapira et al. 2009) and in the somatotropin release-
inhibiting factor (SRIF) system of the temporal cortex of
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-011-1210-x) contains supplementary
material, which is available to authorized users.
A. Minelli (&) ꢀ C. Conte
Dipartimento Medicina Sperimentale Scienze Biochimiche,
`
Sezione Biochimica Cellulare, Universita di Perugia,
Via del Giochetto, 06124 Perugia, Italy
e-mail: aminelli@unipg.it
I. Cacciatore ꢀ C. Cornacchia ꢀ F. Pinnen
Dipartimento di Scienze del Farmaco,
`
Universita ‘‘G. D’Annunzio’’, Via dei Vestini 31,
66100 Chieti, Italy
123

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A. Minelli et al.
Ab25-35-treated rats (Burgos-Ramos et al. 2009). Parkin-
son’s disease (PD), a common neurodegenerative disorder
with a complex aetiology, is characterized by a massive
loss of dopaminergic neurons in the substantia nigra pars
compacta (SNpc) that is responsible for neurological
sequelae (Shapira et al. 2009). Although the cause of
selective dopaminergic neuronal degeneration in PD has
still not been resolved, initiation and progression of the
disease seem to be linked to oxidative stress, closely
related to decreased mitochondrial function and ubiquitin
proteasome system dysfunction (Branco et al. 2010). To
date, 3,4-dihydroxyphenyl-L-alanine (L-dopa, LD) is the
most effective medication for controlling PD symptoms,
particularly those related to bradykinesia (Nagatsua and
Sawadab 2009). Although still a controversial issue
(Weiner 2006), LD itself may exacerbate PD via oxidative
stress. Its auto-oxidative potential from a catechol to a
quinone can generate reactive oxygen species (ROS)
leading to neuronal damage and/or apoptotic or non-
apoptotic cell death (Du et al. 2009). Therefore, the inhi-
bition of oxidation of dopamine (DA) and the inhibition of
ROS formation, both leading to chronic detrimental neur-
oinflammation, are important strategies for preventing the
neurodegenerative disease and for neuroprotective therapy.
The multitarget-directed drug design strategy has already
been used to obtain promising multifunctional drugs for
the treatment of PD and Alzheimer’s disease (AD). Several
dual acting drugs in which LD/DA are linked covalently to
an antioxidant molecule provided sustained release in rat
striatum and seemed to protect against the oxidative stress
deriving from autoxidation of DA (Pinnen et al. 2007,
2009). We showed that N-acetyl-L-methionyl-L-dopa-
methyl ester (Ac-Met-LD-OMe), a dual acting drug in
which LD is linked to methionine, successfully spares LD-
induced neuronal loss in a Parkinson animal model (Mi-
nelli et al. 2010). Given the fact that systemic GPE,
although improving the long-term Parkinsonian motor
deficit in 6-OHDA injured rats did not prevent the loss of
dopamine neurons in the SNpc and striatum (Krishna-
murthi et al. 2004), we hypothesized that a co-drug in
which LD is covalently linked to GPE may represent a
valuable therapeutic improvement in the delivery of LD to
Parkinsonian patients. To address this issue, we examined
the effect of LD-GPE on an in vivo progressive chronic PD
animal model generated by chronic administration of low
doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) that represents a preferential counterpart of idio-
pathic PD (Bezard et al. 1997a, b). By immunohisto-
chemical analyses and studies of transcription factors
activity, we showed that systemic administration of LD-
GPE reduces the oxidative stress and the inflammatory
response thus attenuating the factors underlying neurode-
generative disorders.
Materials and methods
Materials
All the reagents, unless otherwise stated, were from Sigma-
Aldrich (St. Louis, MO, USA). All the antibodies, unless
otherwise stated, were from Santa Cruz Biotech (Santa
Cruz, CA, USA).
Synthesis of Ac-LD(Ac)2-Gly-Pro-Glu(OMe)-OMe
(LD-GPE)
Triethanolamine (TEA) (1.14 ml, 8.17 mmol) and isobutyl
chloroformate (IBCF) (1.08 ml, 8.17 mmol) were added
under stirring to an ice-cold solution of Ac-LD(Ac)2-OH
(2.64 g, 8.17 mmol) in dry dimethylformamide (DMF).
After 15 min at 15°C, H-Gly-Pro-Glu(OMe)-OMe (3.62 g,
8.17 mmol) in dry DMF was added to the mixture at 15°C
with stirring. After 3 h at 0°C and 16 h at 5°C, the reaction
mixture was filtered and the resulting solution was evap-
orated under vacuum. The residue was taken up in AcOEt
and the organic layers were washed with 1 N KHSO4,
saturated aqueous NaHCO3, and brine. The residue
obtained after drying and evaporation was chromato-
graphed on silica gel with CHCl3/MeOH (97:3) as eluant to
give 1.74 g of LD-GPE (1, MW 634.25). Structure and
spectra of LD-GPE were determined by full analysis of the
1H- and 13C-NMR and mass spectral data; homogeneity
was confirmed by TLC on silica gel Merck 60 F254
.
Solutions were routinely dried over anhydrous sodium
sulphate prior to evaporation. Chromatographic purifica-
tions were performed by Merck 60 70-230 mesh ASTM
silica gel column. 1H- and 13C-NMR spectra were recorded
on a Varian VXR 300-MHz spectrometer. Chemical shifts
are reported in parts per million (d) downfield from the
internal standard tetramethylsilane (Me4Si). The LC–MS/
MS system consisted of an LCQ (Thermo Finnigan) ion
trap mass spectrometer (San Jose, CA, USA) equipped with
an electrospray ionization (ESI) source. The capillary
temperature was set at 300°C and the spray voltage at
4.25 kV. The fluid was nebulized using nitrogen (N2) as
both the sheath gas and the auxiliary gas.
Analytical data of LD-GPE: Synthesis yield 33%;
Rf = 0.22, CHCl3/MeOH (97:3); 1H NMR (CDCl3) d
1.90-2.12 (4H, m, Pro b-CH2 and Glu b-CH2), 2.01 (3H, s,
Ac), 2.10–2.17 (2H, m, Glu c-CH2), 2.25 and 2.28 (6H,
29 s, Ac), 2.29–2.40 (2H, m, Pro c-CH2), 3.05–3.12 (2H,
m, LD b-CH2), 3.31–3.50 (2H, m, Pro d-CHA), 3.59–3.65
(2H, m, Pro d-CHB), 3.61 (3H, s, OMe), 3.73 (3H, s,
OMe), 3.80–4.10 (2H, m, Gly a-CH2), 4.49 (2H, m, Pro
a-CH and Glu a-CH), 4.80 (1H, m, LD a-CH), 6.45 (1/2 H,
t, LD NH), 6.61 (1/2 H, t, LD NH), 6.90–7.12 (4H, m, ArH
and Gly NH), 7.30 (1/2 H, d, Glu NH), 7.35 (1/2 H, d, Glu
123

Molecular mechanism underlying the cerebral effect of Gly-Pro-Glu
1361
NH). 13C NMR (CDCl3) d 20.83 and 20.87 (29 Ac),
23.14 (Ac), 24.93 (Pro b), 27.12 (Glu b), 28.26 (Pro c),
30.31 (Glu c), 37.77 (LD b), 42.25 (Gly a), 46.76 (Pro d),
52.02 (OCH3), 52.27 (LD a), 52.78 (OCH3), 54.06 (Glu a),
60.37 (Pro a), 123.61–142.05 (Ar), 168.06, 168.64, 169.03,
170.80, 171.12, 171.28, 172.27, 173.66 (89 CO). MS
(ESI) m/z 634 (M–H).
with dithionitrobenzoic acid (DTNB) at 412 nm (molar
extinction coefficient 13.6 mM-1 cm-1) as described
(Rahman et al. 2006).
Real time PCR
Striatum RNA was isolated with TRIZOL Reagent (Invit-
rogen Ltd., Paisley, UK) according to the manufacturer’s
instructions and cDNA was synthesised using iScript
cDNA synthesis kit (Bio-Rad Lab, Hercules, CA, USA)
Real time PCR was performed using the iCycler iQ
detection system (Bio-Rad) and SYBR Green chemistry.
Mouse primer sequences, obtained from Invitrogen (Invit-
rogen Ltd., Paisley, UK) are listed in Table S1.
Animals
Two month-old male C57BL/6 mice (22–25 g) were
housed at the Laboratory Animal Research Centre of Pe-
rugia University. The animals were fed ad libitum and
maintained at a constant temperature of 24°C and 12-h
light/dark cycle.
SYBR Green RT-PCR amplifications were carried out in
a 96-well plate in a 25 ll reaction volume that contained
12.5 ll of 29 iQTM SYBRÒ Green SuperMix (Bio-Rad),
400 nM forward and reverse primers, and 5–40 ng of
cDNA. In each assay, no-template controls were included
and each sample was run in triplicates. The thermal profile
consisted of incubation at 95°C for 3 min, followed by 40
cycles of denaturation for 10 s at 95°C and an annealing/
extension step of 30 s at 62°C. Mean of Ct values of the
stimulated sample was compared to the untreated control
sample. DCt is the difference in Ct values derived from the
target gene (in each assayed sample) and GAPDH, while
DDCt represents the difference between the paired samples.
Ethics statement
All experimental procedures were carried out in accor-
dance with European Directives, approved by the Institu-
tional Animal Care and Use Committee of Perugia
University (N33/2009). Efforts were made to minimise
animal number, stress/discomfort.
Animal treatments
Progressive chronic PD animal model was generated with
low doses of MPTP for 20 days (Bezard et al. 1997a, b;
Schmidt and Ferger 2001). Thirty animals were divided in
four groups as follows: (1) one group (6 animals) was
treated with saline for 20 days (control), (2) one group (8
animals) was treated with daily (0900 hours) injections of
MPTP hydrochloride (4 mg/kg, i.p., Toronto Research
chemicals Inc., Canada) in saline for 20 days, (3) one
group (8 animals) was treated with daily (0900 hours)
injections of MPTP hydrochloride (4 mg/kg, i.p.) and LD
(100 mg/kg, i.p), (4) one group (8 animals) was treated
with daily (0900 hours) injections of MPTP hydrochloride
(4 mg/kg, i.p.) and LD-GPE (254 mg/kg, i.p). Neither
body weight changes nor mortality were observed during
treatments. All the animals were killed at the end of the
treatments. One hemisphere was processed for immuno-
histochemistry and the isolated striatum was used for other
determinations.
The n-fold differential ratio was expressed as 2-DDCt
.
Tyrosine hydroxylase and glial fibrillar acidic protein
immunohistochemistry
Immediately after killing, brains were removed and one
hemisphere was immersion-fixed in Carnoy solution and
embedded in paraffin. Coronal sections (10 lm) were
obtained from the frontal to the caudal pole. Cerebral
sections at defined levels were processed to assess immu-
noreactivity for TH and GFAP (Minelli et al. 2010).
Briefly, sections were sequentially incubated with: (1) 20%
normal horse serum in phosphate buffered saline (PBS) for
20 min; (2) anti-TH and anti-GFAP (DakoCytomation,
Denmark) antibodies (1:100) in PBS containing 20% nor-
mal serum and 0.03% Triton X-100 overnight at 4°C; (3)
biotinylated secondary antibody (1:40, Vector Laborato-
ries, Burlington, Ontario, CA, USA) for 60 min; (4) avi-
din–biotin peroxidase complex (Vectastain ABC Kit,
Vector Labs, Ontario, CA, USA) for 45 min. The reaction
product was visualized by incubation with H2O2 and 3-30-
diaminobenzidine (DAB) and slices analysed by Olympus
B950 system microscope. Immunohistochemical reactivity
was semi-quantified by analysing photomicrographs with
ImageJ software (National Institute of Health).
Glutathione determination
Fresh striatum samples, washed in ice-cold isotonic saline
solution, were homogenized with 0.6% sulfosalicylic acid/
0.01 Triton X-100 on ice to remove proteins, and centri-
fuged at 10,0009g for 10 min. The supernatant was
immediately used for glutathione (GSH) determination
123

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A. Minelli et al.
Subcellular fractionation
Results
Striatum samples were washed three times with PBS on
ice, homogenised in PBS, and collected by centrifugation
for 5 min at 5009g. Pellets were then processed with NE-
PERÒ Nuclear and Cytoplasmic Extraction Reagents
(Pierce Biotechnology, Rockford, IL, USA) according to
manufacturer’s instruction. Protein concentrations of
nuclear and cytoplasmic fractions were determined by Bio-
Rad protein assay kit (Bio-Rad).
Chemistry
Ac-LD(Ac)2-Gly-Pro-Glu(OMe)-OMe (LD-GPE, 1, MW
634.25) was synthesized by solution phase procedures
through the coupling of Ac-LD(Ac)2-OH (2) with H-Gly-
Pro-Glu(OMe)-OMe (3) (Brimble et al. 2005). Compound
2 was obtained after N-acetylation of H-LD(Ac)2-OH
(Bodor et al. 1977; Dahmer and Whitesides 1989) (Fig. 1).
Western blotting analyses
LD-GPE reduces MPTP neurotoxicity and gliosis
Striatum nuclear/cytosolic extracts (60 lg) were loaded on
12% SDS-PAGE and protein levels determined by Western
blotting using appropriate antibodies Nrf2 (C-20), NF-jB
p65(A), inducible nitric oxide synthase (iNOS,C-11),
1:200; heme oxygenase 1 (HO-1, C-18), 1:50). Lamin B(H-
90) and b-actin (I-19), antibodies (1:400) were used as
marker proteins for nuclear/cytosolic extracts. After sec-
ondary incubation in horseradish peroxidase-conjugated
anti IgG antibody (1:5,000) (Amersham Bioscience, Little
Chalfont, UK), the immunocomplexes were visualized with
an enhanced chemiluminescence kit (ECL, Pierce Bio-
technology, Rockford, IL, USA). Bands were analysed
with the Bandscan software.
Systemic administration of MPTP to C57BL/6 mice and
the subsequent degeneration of dopaminergic neurons in
the substantia nigra can produce striking similarities to PD
diagnosed in humans (Schmidt and Ferger 2001). The
schedule of MPTP administration used in the study pro-
vides a progressive chronic PD model, successfully used
for studies on neuroprotection and compensatory mecha-
nisms (Schmidt and Ferger 2001). We analysed MPTP-
induced neurotoxicity using TH immunoreactivity as a
phenotypic marker for dopaminergic neurons (Fig. 2a).
Immunostaining for TH-containing dopaminergic neurons
in the midbrain of chronically treated animals showed a
decline in TH-immunoreactivity. Semi-quantitative analy-
ses showed that the significant reduction (55 7%) in TH-
density by the end of the chronic MPTP administration was
attenuated by treatment with LD and LD-GPE (78 9 vs.
89 8%). Real time PCR analyses of the expression of
TH gene showed that TH gene expression was restored to
control value only by LD-GPE treatment (Fig. 2b). Given
that MPTP administration causes a reactive gliosis (Inna-
morato et al. 2010), we verified whether LD-GPE was also
capable of attenuating MPTP-inflammatory effects by
GFAP immunoreactivity (Fig. 2a). Treatment with LD-
GPE nearly restored the resting morphology. Real time
PCR analyses of each striatum were used to quantify
mRNA levels of astrocyte GFAP (glial fibrillar acidic
protein), microglia Iba-1 (ionized calcium binding adaptor
molecule 1), and brain macrophages CD11b (integrin aM
chain), as markers of inflammation (Rojo et al. 2010). LD-
GPE treatment resulted in a marked down-regulation of the
expression of GFAP and CD11b, showing a reduced
Striatal dopamine and 1-methyl-4-phenylpyridinium
levels
Striatal DA levels were individually determined by
HPLC-EC assay, as described (Pinnen et al. 2007).
1-methyl-4-phenylpyridinium (MPP?) levels were indi-
vidually determined by HPLC–UV, as described (Andrews
et al. 2005). Concentration of DA/MPP? were expressed as
nanogram/milligram of striatum.
Statistical analysis
Each assay was performed at least in triplicate unless
otherwise indicated. Data are expressed as mean val-
ues SD, and significance was assessed by a one-way
ANOVA followed by Tukey’s test. A P value \0.05 was
considered significant in all cases.
Fig. 1 Synthesis of LD-GPE.
Reagents and conditions:
Isobutyl chloroformate (IBCF),
triethanolamine (TEA), and
dimethylformamide (DMF), 3 h
at 0°C, and then 15 h at 4°C
123

Molecular mechanism underlying the cerebral effect of Gly-Pro-Glu
1363
Fig. 2 LD-GPE protects from dopaminergic neuronal death in mice
Parkinson’s model. MPTP hydrochloride (4 mg/kg, dissolved in
saline) was injected daily i.p. for 20 days to male mice (C57BL/6
mice, 8 weeks old). Animals were divided in four groups (6–8 mice)
and treated with MPTP (4 mg/kg), MPTP plus LD (100 mg/kg), and
MPTP plus LD-GPE (254 mg/kg). Control mice were treated with
saline for 20 days and killed after the last injection. After killing,
brains were sagittally divided and hemispheres used for immunohis-
tochemical analyses. a TH- and GFAP-stained sections of brain.
Strong decline in TH immunoreactivity was detected after MPTP
treatment. Mice treated with LD-GPE showed an increased TH-
density and glial resting morphology. One representative experiment
is shown; b real time PCR analyses showing expression of TH and
inflammatory genes in the isolated striatum from each animal.
Changes in gene expression were normalized to GAPDH expression
and presented as 2-DDCt. Relative mRNA abundance of each gene in
striatum of untreated animals was assumed to be 1.0 (control);
c striatal DA levels determination. Each striatum was used for
determinations by HPLC-EC. Dopamine levels are given as percent-
age of control (100% control: 15 4 ng/mg of striatum). Data
represent mean values SD of six/eight experiments. *P \ 0.05
#
versus control striata, P \ 0.05 versus MPTP-treated striata
inflammatory response to MPTP treatment. LD treatment
did not significantly decrease GFAP and Iba-1 mRNA
levels (Fig. 2b). Striatal dopamine confirmed that periph-
eral LD-GPE administration has central effects (Fig. 2c).
MPP? levels (2.24 0.7 ng/mg of striatum) of MPTP-
treated animals were not significantly modified by LD and
LD-GPE treatment (2.10 0.5 vs. 2.18 0.6), showing
that LD-GPE does not interfere with MPTP access.
LD-GPE increases Nrf2 nuclear translocation
GPE administration to Ab25-35-treated rats is coincident
with activation of protective pathways via cAMP response
element-binding (CREB) activation (Burgos-Ramos et al.
2009). CREB is a transcription factor closely related to
Nrf2 and NF-jB (Katoh et al. 2001; Zou and Crews 2006).
To investigate whether and how LD-GPE interferes with
the activation/suppression of Nrf2 and NF-jB, we analysed
by Western blotting the nuclear translocation of these
transcription factors, responsible for the integrated tran-
scriptional responses to cellular stressors (Brigelius-Flohe
Fig. 3 LD-GPE increases Nrf2 nuclear translocation while reducing
NF-jB. Nuclear extracts were obtained from striatum of each animal
and used for Western blotting analysis. 60 lg of proteins were
immunodetected with the indicated antibodies. Anti-lamin B and anti-
b-actin antibodies were used as markers for nuclear and cytosolic
extracts. One representative experiment is shown. Data represent
mean values SD of six/eight experiments. *P \ 0.05 versus
#
control striata, P \ 0.05 versus MPTP-treated striata
123

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A. Minelli et al.
`
and Flohe 2011).We found that MPTP treatment resulted in
a significantly increased NF-jB nuclear translocation
accompanied by a modest Nrf2 translocation whereas LD-
GPE treatment caused a marked translocation of Nrf2
while suppressing NF-jB. LD treatment did not signifi-
cantly modify NF-jB nuclear translocation compared to
MPTP treatment, although increasing the nuclear translo-
cation of Nrf2, confirming that LD interferes with cellular
redox homeostasis (Fig. 3). Analyses of gene expression
by real time PCR confirmed the suppression of the NF-jB
signalling and the activation of the Nrf2-antioxidant
response by LD-GPE treatment (Fig. 4a). We found a
significant up-regulation of the expression of Nrf2-driven
genes, responsible for endogenous cellular defence and a
down-regulation of NF-jB-driven genes, responsible for
inflammatory responses. These findings indicate that the
beneficial effects of LD-GPE depend on the simultaneous
regulation of Nrf2 and NF-jB within striatum. We next
decided to assess the efficiency of the transcriptional reg-
ulation of these two transcription factors, which, in addi-
tion to the nuclear translocation of protein components, is
of paramount importance for the efficiency of the NF-jB
and Nrf2 systems (Salminen et al. 2008). Determination of
protein expression levels of heme oxygenase 1 (HO-1) and
inducible nitric oxide synthase (iNOS) confirmed the
efficiency of the transcriptional regulation of these tran-
scription factors (Fig. 4b). Striatal glutathione (GSH) level
determinations were compatible with the efficacy of the
co-drug treatment that restored control GSH levels
(Fig. 4c).
Fig. 4 LD-GPE up-regulates
Nrf2-antioxidant while
suppressing NF-jB-pro-
inflammatory genes. a Real time
PCR analyses of mRNA level of
Nrf2- and NF-jB-driven genes.
Changes in gene expression
were normalized to GAPDH
expression and presented as
2
-DDCt. Relative mRNA
abundance of each gene in
untreated striata was assumed to
be 1.0 (control); b Western
blotting analyses of HO-1 and
iNOS. 60 lg of cytosolic extract
were immunodetected with the
indicated antibodies and b-actin
used as loading control. One
representative experiment is
shown; c striatum GSH content
(100% control GSH:
278 17 nmol/mg protein).
Data represent mean
values SD from six/eight
animal per group from two
separate experiments.
*P \ 0.05 versus control striata;
#P \ 0.05 versus MPTP-treated
striata
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Molecular mechanism underlying the cerebral effect of Gly-Pro-Glu
1365
`
and inflammatory stressors (Brigelius-Flohe and Flohe
Discussion
2011). In this study, by investigating in our animal models
the role of these transcription factors and their cellular
outcomes, we showed that both transcription factors sense
and respond to cell stress and that their simultaneous reg-
ulation dictates the cellular response. Striata of animals
treated with MPTP showed a marked nuclear translocation/
activation of NF-jB and a more moderate increase in Nrf2
nuclear levels, resulting in a significant up-regulation of
NF-jB-driven genes and a modest induction of Nrf2-driven
genes. This mutual modulation resulted in a marked cell
loss in the MPTP treatment (Bezard et al. 1997a, b).
Treatment with LD-GPE suppressed NF-jB signalling
while strongly activating Nrf2, confirming that the induc-
tion of Nrf2-mediated antioxidant enzymes and the
reduction of NF-jB-mediated inflammatory mediators/
enzymes exert beneficial effects (Jung et al. 2010; Minelli
et al. 2009). In LD-GPE-treated animals, glial reactivity
was reduced while striatum GSH levels and HO-1
expression/protein levels restored/increased, resulting in a
neuroprotective effect. Nrf2 activation and HO-1 activity
contribute to the anti-inflammatory activity of cells and
tissues (Innamorato et al. 2010; Rojo et al. 2010; Chen
et al. 2009; Jadhav et al. 2008; Lee and Chau 2002; Jung
et al. 2001), thus an increased expression of antioxidant
genes via Nrf2 activation might be a feasible therapeutic
target to reduce neuronal death and inflammation in PD
patients. Here we provided the first evidence that the dual
acting drug LD-GPE induces the expression of HO-1 via
Nrf2 activation in animal striatum while suppressing the
expression of NF-jB-driven genes (Scheme 1) thus
Two major observations are reported in this study. First, we
showed that the new dual acting drug LD-GPE has
potential application in PD therapy, as confirmed by
immunohistochemical and real time PCR analyses; second,
by analysing the mechanism underlying the beneficial
effects of LD-GPE, we showed the simultaneous regulation
of Nrf2 and NF-jB, and, more importantly, the cellular
consequences of such co-regulation.
Parkinson’s disease is the most common neurodegen-
erative movement disorder whose symptoms, in the
beginning, can be successfully treated with LD (Schapira
2009; Nagatsua and Sawadab 2009). In PD, loss of striatal
dopaminergic terminals and degeneration of DA neurons in
the substantia nigra (SN) are associated with glial reactions
that, in turn, cause an inflammation-derived oxidative
stress. LD and its metabolite dopamine (DA) can enhance
oxidative stress and accelerate the degenerative process of
residual cells in patients with PD (Weiner 2006; Hattoria
et al. 2009). LD itself may contribute to the progression of
PD via oxidative stress: its auto-oxidative potential from a
catechol to a quinone can generate reactive oxygen species
(ROS) leading to neuronal damage and/or apoptotic or non-
apoptotic cell death (Du et al. 2009). Therefore, activation
of antioxidant responses and inhibition of inflammatory/
apoptotic pathways are important strategies for preventing
the neurodegenerative disease and for neuroprotective
therapy. Here, LD was bound to GPE, the N-terminal tri-
peptide of insulin-like growth factor-I. Data from the lit-
erature (Sizonenko et al. 2001; Saura et al. 1999; Alexi
et al. 1999; Guan et al. 1999, 2000, 2004; Aguado-Llera
et al. 2004; Zhao et al. 2005; Shapira et al. 2009; Krish-
namurthi et al. 2004; Burgos-Ramos et al. 2009) indicate
that peripheral GPE administration can have central neu-
roprotective effect, even though it does not prevent the loss
of tyrosine hydroxylase in the SNpc and the striatum
(Krishnamurthi et al. 2004). Thus, we investigated the
therapeutic efficacy of LD-GPE in a progressive chronic
PD animal model. We found that the toxicity of MPTP
treatment was significantly reduced by LD-GPE by
immunohistochemical and real time PCR analyses. mRNA
levels of TH and inflammation marker genes were restored
to control values by the co-drug. The marked anti-inflam-
matory effect of LD-GPE was not unexpected since GPE,
by binding to glial cells, leads to a reduced release of
neurotoxic products (Baker et al. 2005; Sizonenko et al.
2001; Guan et al. 2004). GPE administration to Ab25-
35-treated rats activates protective pathways via CREB
activation (Burgos-Ramos et al. 2009), which, in turn, is
closely related to Nrf2 and NF-jB modulation (Katoh et al.
2001; Zou and Crews 2006). The interplay between Nrf2
and NF-jB is responsible for cellular responses to chemical
Scheme 1 LD-GPE is neuroprotective against MPTP neurotoxicity
in mice via Nrf2/NF-jB modulation
123

1366
A. Minelli et al.
Brimble MA, Trotter NS, Harris PW, Sieg F (2005) Synthesis and
pharmacological evaluation of side chain modified glutamic acid
analogues of the neuroprotective agent glycyl-L-prolyl-L-glu-
tamic acid (GPE). Bioorg Med Chem 13:519–532
holding great potential for the therapeutic strategies to PD
treatment. Indeed, although in our study the co-drug was
administered with the neurotoxin, a common pitfall of
animal models, our results showed a therapeutic superiority
of LD-GPE over LD, which, to date, is the most effective
medication for controlling PD symptoms. We also showed
an inverse correlation between Nrf2 and NF-jB, not found
in the LD treated animals, that might explain the observed
reduction in glial activation, thus supporting the proposal
that increased expression of antioxidant genes via Nrf2
activation might be a feasible therapeutic target to reduce
neuronal death and inflammation in PD patients.
´
Burgos-Ramos E, Martos-Moreno GA, Lopez MG, Herranz R, Aguado-
Llera D, Egea J, Frechilla D, Cenarruzabeitia E, Leon R, Arilla-
´
Ferreiro E, Argente J, Barrios V (2009) The N-terminal tripeptide
of insulin-like growth factor-I protects against beta-amyloid-
induced somatostatin depletion by calcium and glycogen synthase
kinase 3 beta modulation. J Neurochem 109:360–370
Chen PC, Vargas MR, Pani AK, Smeyne RJ, Johnson DA, Kan YW,
Johnson JA (2009) Nrf2-mediated neuroprotection in the MPTP
mouse model of Parkinson’s disease: critical role for the
astrocyte. Proc Natl Acad Sci USA 106:2933–2938
Dahmer J, Whitesides GM (1989) Kinetic resolution of unnatural
and rarely occurring amino acids: enantioselective hydrolysis of
N-acyl amino acids catalyzed by acylase I.
111:6354–6364
Du F, Qian ZM, Zhu L, Wu XM, Yung WH, Tsim TY, Ke Y (2009)
L-dopa neurotoxicity is mediated by up-regulation of DMT1-IRE
expression. PLoS One 4:e4593
J Am Soc
Acknowledgments The authors thank Mary Kerrigan (MA, Cantab)
for helpful linguistic suggestions. The study was funded by Fondaz-
ione Cassa di Risparmio, Perugia, Italy (Grant 2009-010-0437). The
funder had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Guan J, Waldvogel HJ, Faull RL, Gluckman PD, Williams CE (1999)
The effects of the N-terminal tripeptide of insulin-like growth
factor-1, glycine–proline–glutamate in different regions follow-
ing hypoxic-ischemic brain injury in adult rats. Neuroscience
89:649–659
Conflict of interest The authors declare no conflict of interest.
Guan J, Krishnamurthi R, Waldvogel HJ, Faull RL, Clark R,
Gluckman P (2000) N-terminal tripeptide of IGF-1 (GPE)
prevents the loss of TH positive neurons after 6-OHDA induced
nigral lesion in rats. Brain Res 859:286–292
Guan J, Thomas GB, Lin H, Mathai S, Bachelor DC, George S,
Gluckman PD (2004) Neuroprotective effects of the N-terminal
tripeptide of insulin-like growth factor-1, glycine–proline–
glutamate (GPE) following intravenous infusion in hypoxic–
ischemic adult rats. Neuropharmacology 47:892–903
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