Mol Neurobiol (2018) 55:201–212
DOI 10.1007/s12035-017-0733-x
Deleterious Effects of VEGFR2 and RET Inhibition
in a Preclinical Model of Parkinson’s Disease
C. Requejo 1 & J. A. Ruiz-Ortega 2 & H. Bengoetxea 1 & S. Bulnes 1 & L. Ugedo 2 &
J. V. Lafuente 1,3,4
Published online: 24 August 2017
# Springer Science+Business Media, LLC 2017
Abstract Neurotrophic factors (NTFs) are a promising thera-
peutic option for Parkinson’s disease (PD). They exert their
function through tyrosine kinase receptors. Our goal was to
assess the effects of administering a selective tyrosine kinase
inhibitor (vandetanib) that blocks VEGFR2 and RET receptors
in a preclinical model of PD. Rats underwent intrastriatal injec-
tions of 6-hydroxydopamine (6-OHDA). Two weeks later, the
rats received 30 mg/kg vandetanib or saline orally. The effects
were assessed using the rotational behavioral test, tyrosine
hydroxylase (TH) immunohistochemistry, and western blot. In
6-OHDA-lesioned rats, motor symptoms were almost undetect-
able, but morphological and biochemical changes were signifi-
cant. Vandetanib treatment, combined with the presence of
6-OHDA lesions, significantly increased behavioral impairment
and morphological and biochemical changes. Therefore, after
vandetanib treatment, the TH-immunopositive striatal
volume, the percentage of TH+ neurons, and the extent of the
axodendritic network in the substantia nigra decreased. Glial
fibrillary acidic protein-positivity significantly decreased in the
striatum and substantia nigra in the vandetanib-treated group. In
addition, p-Akt and p-ERK 1/2 levels were significantly lower
* C. Requejo
catalina.requejo@ehu.eus
1
LaNCE, Department of Neuroscience, University of the Basque
Country (UPV/EHU), Vizcaya, Leioa, Spain
2
Department of Pharmacology, University of the Basque Country
(UPV/EHU), Vizcaya, Leioa, Spain
3
Nanoneurosurgery Group, BioCruces Health Research Institute,
48903 Barakaldo, Bizkaia, Spain
4
Faculty of Health Science, Universidad Autónoma de Chile, Santiago
de Chile, Chile
and caspase-3 expression significantly increased after vandeta-
nib administration. In conclusion, we demonstrate for the first
time the deleterious effect of a tyrosine kinase inhibitor on the
dopaminergic system, supporting the beneficial and synergistic
effect of NTFs reported in previous papers.
Keywords Parkinson’s disease . 6-OHDA . Preclinical
model . Vandetanib . VEGFR2 . RET . Neurotrophic factors
Introduction
Parkinson’s disease (PD) is a complex and heterogeneous
disease that involves multiple pathological mechanisms,
inducing cell death [1]. Several neurotrophic factors (NTFs)
have been shown to protect dopaminergic neurons and glial
cells against excitotoxicity induced by the activation of
specific signaling pathways that are responsible for cell sur-
vival and axonal sprouting [2, 3].
Glial cell line-derived neurotrophic factor (GDNF) mediates
its action through a complex signaling network. In fact, the
GDNF receptor complex consists of the RET receptor tyrosine
kinase and glycosylphosphatidylinositol (GPI)-linked GDNF
family receptor α1 (GFRα1) [4]. RET is required for the
long-term survival of nigral dopamine neurons and the mainte-
nance of their striatal innervation in mice [5, 6]. A recent study
that used an experimental model of PD demonstrated that GDNF
did not display a neurorestorative or neuroprotective effect after
suppression of RET in dopaminergic neurons [6]. GDNF/RET
signaling initiates upon binding of the GDNF dimer to GFRα1
receptors linked to the plasma membrane through a
glycosylphosphatidylinositol anchor [4]. Dimerization of RET
triggers its autophosphorylation to initiate various intracellular
signaling cascades, such as the phosphoinositide 3-kinase
(PI3K)/Akt and the extracellular signal-regulated kinase
202
(ERK)/mitogen-activated protein (MAP) kinase pathways,
among others. These pathways regulate cell survival, prolifera-
tion, differentiation, neurite outgrowth, synaptic plasticity, and
morphogenesis [4].
Vascular endothelial growth factor (VEGF) is an important
mediator of angiogenesis and is also involved in cell survival
and proliferation [7–9]. VEGF isoforms bind to three different
receptors (VEGFR1, VEGFR2, and VEGFR3). The neuropro-
tective effects are predominantly mediated by VEGFR-2 [10,
11] via the PI3K/Akt and MEK/ERK pathways [12, 13].
Numerous studies support the capacity of VEGF to display
neuroprotection and/or neurorescue on dopaminergic neurons
in both in vitro and in vivo models of PD [14, 15].
Some findings support the synergy between VEGF and
GDNF. Therefore, the combined administration of VEGF
and GDNF has been demonstrated as a neuroregenerative
strategy to recover dopaminergic populations of neurons in
PD and to preserve dopaminergic terminals in experimental
animal models [16–19].
Vandetanib (ZD6474) is a reversible, orally bioavailable,
tyrosine kinase inhibitor (TKI) that targets RET, VEGFR2,
and VEGFR3, as well as EGFR at higher concentrations
[20]. Vandetanib competes with ATP binding in the catalytic
domain of several tyrosine kinase receptors, such as VEGFR2
and RET, to inhibit autophosphorylation [21]. As a therapeutic
strategy, this drug is primarily administered to patients with
advanced or metastatic medullary thyroid cancer (MTC) [22].
Therefore, its administration in neurodegenerative disorders is
not of therapeutic interest, but its use in the elucidation of the
role of NTFs in signaling pathways is of interest.
The goal of the present study was to assess the role of
constitutive VEGF and GDNF in the maintenance and recov-
ery of the nigrostriatal pathway in a preclinical model of PD.
The inhibition of VEGF and GDNF signaling could help to
identify which downstream signaling cascades are involved in
the achievement of the neurorestorative and neuroprotective
effects that have been reported for both neurotrophic factors.
Materials and Methods
The experiments were performed on 16 male Sprague-Dawley
rats that weighed 275–320 g. The animals were housed under
normal laboratory conditions. All animal procedures were car-
ried out in accordance with the Ethical Committee and Animal
Welfare (CEBA) of the University of the Basque Country
(CEBA/154/2010//RUIZ ORTEGA) and in agreement with
Spanish Royal Decree RD 1201/2005, European Directive
2003/65/EC, and the European Recommendation 2007/526/
EC on the protection of animals used for scientific purposes.
All rats received 6-hydroxydopamine (6-OHDA) injec-
tions into the right striatum. After 2 weeks, they were divided
into two groups: the control group (n = 8 rats) that received
Mol Neurobiol (2018) 55:201–212
only saline solution orally and the treated group (n = 8) that
received 30 mg/kg/day of vandetanib orally for 1 week (Ref:
V-9402, LC Laboratories, USA).
The amphetamine-induced behavior test was performed dur-
ing the 2nd and 3rd weeks after lesion induction. Then, the rats
were sacrificed, and the brains were processed for histology
(four animals per group) or western blot (four animals per
group). 6-OHDA lesions were induced as described in previous
studies [17, 18]. Ipsilateral rotations were recorded every 5 min
for 90 min after the i.p. administration of the amphetamine
(5 mg/kg) (Sigma, St. Louis, USA) with an automated rotame-
ter (multicounter LE3806; Harvard Apparatus, Holliston, MA,
USA). The data are expressed as turns per minute (tpm). The
preclinical stage was characterized by the mild presence of
motor symptoms, no more than 1–3 tpm.
Tissue Processing for Histology and Stereological Analysis
The animals were anesthetized with chloral hydrate (400 mg.
kg−1, i.p.) (Ref: 141,975, Panreac Quimica SA, Barcelona,
Spain) and transcardially perfused with 0.9% NaCl followed
by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered
saline (PBS). The brains were removed, postfixed, and later
transferred to a cryopreservative solution (30% sucrose, PBS
0.1 M). Coronal brain slices were obtained with a freezing
cryotome (50 μm thick) and collected in PBS containing
0.6% sodium azide for storage.
Double immunofluorescence was carried out on free-floating
sections to detect caspase-3. Briefly, the sections were incubated
overnight at 4 °C in a blocking solution [5% bovine serum albu-
min (BSA) and 0.05% Triton X-100 in 0.1 M PBS] with a
cocktail of primary antibodies containing rabbit anti-caspase 3
(H-277) (Ref: sc-7148, Santa Cruz Biotechnology Inc., Spain;
1:50) and monoclonal mouse anti-NeuN (Ref: MAB377,
Chemicon International, Inc., Spain; 1:100) as a neuronal marker.
After washing, the sections were further incubated in a blocking
solution containing Alexa 488 conjugate anti-mouse IgG (Ref:
A11029; Invitrogen; 1:400) and Alexa 568 conjugate goat
anti-rabbit IgG (Ref: A11036 Invitrogen; 1:400) for 1 h at room
temperature in darkness.
Hoechst 33258 was added to counterstain the nuclei for
10 min. The slices were washed, mounted, and coverslipped
with the medium, Vectashield (Ref: x-0517; Vector laborato-
ries). Images were examined under an Olympus Fluoview
FV500 confocal microscope using sequential acquisition to
avoid the overlapping of fluorescent emission spectra. The im-
ages were evaluated with FV 10-ASW 1.6 Viewer and Adobe
Creative Suite 4.
Immunohistochemistry for tyrosine hydroxylase (TH) and
glial fibrillary acidic protein (GFAP) was performed on
free-floating slices following a standard avidin-biotin immuno-
histochemical protocol as described previously [17, 19]. To vi-
sualize and evaluate the results of sections stained with HRP-
Mol Neurobiol (2018) 55:201–212
DAB, an Olympus BX-50 photomicroscope was used. For ste-
reology, a computerized image analysis system (Mercator Image
Analysis system, Explora Nova, La Rochelle, France) connected
to the previously described microscope was utilized. The volume
of the caudate-putamen complex (CPC) and the TH-negative
volume of CPC were calculated to assess the lesions as previous-
ly described by Requejo and collaborators [17, 19].
To elucidate the gradient distribution, measurements from
three CPC levels according to Paxinos & Watson’s Atlas were
estimated {rostral (bregma + 0.70 mm), middle (bregma
− 0.26 mm), and caudal (bregma − 0.80 mm) sections [23]}.
TH-immunoreactive (TH-ir) neurons and the axodendritic
network (ADN) were measured using a stereological tool (an
optical fractionator) provided by the computerized analysis sys-
tem. Probes of 50 × 50 μm separated by 100 μm were launched
into the entire substantia nigra (SN) and into the external SN
(eSN) [17]. The ADN density was also analyzed in the
substantia nigra reticulate (SNr). Immunopositive neurons and
the ADN inside the probe or that crossed the right side of the X–
Y axis were counted. TH-ir neurons and ADN were calculated
per section and per animal, with consideration of all SN slices.
Integrated Optical Density
The integrated optical density (IOD) was measured from pic-
tures of coronal sections of the striatum and the SN stained
with GFAP. These sections were digitalized and captured with
a 1200 dpi resolution digital scanner (Epson, Suwa, Japan).
The measurements were completed using Image J software. In
addition, the IOD reading was corrected for background stain-
ing (subtracting the values of a region outside of the tissue).
Optical density values were expressed as the percentage of the
ipsilateral striatum versus the contralateral striatum, which
was considered to be 100%.
Sample Processing for Western Blot
The rats were anesthetized using chloral hydrate (400 mg.kg−1,
i.p.) and then sacrificed. The brains were removed for micro-
dissection of the ipsilateral striatum, the contralateral striatum,
and the SN and quickly frozen. Protein isolation samples were
homogenized in lysis buffer containing a protease inhibitor
cocktail (Ref: P-8340, Sigma-Aldrich, Spain). The lysates were
centrifuged at 4 °C for 15 min (15,500×g). Solubilized proteins
were recovered from the supernatants and quantified using the
Bio-Rad Protein Assay (Ref: 500-0006, Bio-Rad Laboratories
SA, Spain) with BSA as the standard [24].
Western Blot Analysis
Twenty micrograms of total protein per sample was loaded into
polyacrylamide CRITERION TGX 12% gels (Ref: 567-1045,
Bio-Rad Laboratories Inc., Spain) and separated by
203
electrophoresis (Mini-Protean 3 Electrophoresis Cell, Bio-Rad
Laboratories SA). The proteins in the gels were then transferred
to a PVDF membrane (Ref: 170-4157, Transfer Pack Trans-Blot
Turbo, Bio-Rad Laboratories Inc., Spain) in a Trans-Blot Turbo
Transfer System (Bio-Rad, USA) for 7 min. The blots were
incubated in blocking buffer containing 5% BSA and 1% normal
goat serum (Ref: S-1000, Vector Laboratories, USA) in
Tris-buffered saline including 0.1% Tween-20 (TBS-T). Then,
the membranes were incubated overnight at 4 °C with the fol-
lowing primary antibodies: rabbit anti-Phospho-Akt (Ser 473)
(Ref: 9271, Cell Signaling Technology Inc., USA; 1:1000), rab-
bit anti-Akt (Ref: 9272, Cell Signaling Technology Inc., USA;
1:1000), rabbit anti-Phospho-p44/42 MAPK (ERK 1/2) (Thr
202/204) (Ref: 9101, Cell Signaling Technology Inc., EEUU;
1:1000), rabbit anti-P44/42 MAPK (ERK 1/2) (Ref: 9102, Cell
Signaling Technology Inc., EEUU; 1:1000), rabbit anti-caspase
3 (H-277) (Ref: sc- 7148, Santa Cruz Biotechnology Inc., Spain;
1:1000), rabbit anti-β-Actin (Ref: A2066, Sigma-Aldrich, Spain;
1:2000), and rabbit anti-Beta-Tubulin (Ref: NB600-936, Novus
Biologicals, USA; 1:1000). The membranes were then incubated
with anti-rabbit IgG peroxidase conjugate secondary antibodies
(Ref: A-6154, Sigma-Aldrich, Spain; 1:2000). The immunoblots
were visualized with an enhanced chemiluminescence kit (Ref:
RPN 2232, GE Healthcare Life Science, UK). The luminescence
of the reaction product was detected in a personal scanner,
LI-COR C-DiGit (LI-COR, Bonsai Advanced Technologies
SL, Spain), and quantified with Image Studio Lite 4.0 software
(LI-COR, Bonsai Advanced Technologies SL, Spain). β-Actin
and β-tubulin were used as controls.
Statistical Analysis
All values are expressed as the mean ± SE (standard error). The
statistical analysis was performed with SPSS Statistics (v 20;
IBM Corporation, Armonk, NY, USA). Prior to the analysis,
the Shapiro–Wilk test was used to assess the normal distribution
of the samples, and Levene’s test was used to determine the
homogeneity of variance. A one-way analysis of variance
(ANOVA) with Tamhane’s post hoc test was used to explore
the differences between rostro-caudal gradients within each ex-
perimental group. The Student’s t test was used to compare
intergroup differences. Values are considered statistically signif-
icant when P < 0.05.
Results
After 2 weeks, the unilateral 6-OHDA intrastriatal injections
produced an early partial model of PD, with nearly undetect-
able motor symptoms but apparent morphological and bio-
chemical changes. This model allows for the study of the roles
of VEGFR2 and RET in the development of PD using van-
detanib as an inhibitor.
204 Mol Neurobiol (2018) 55:201–212

Behavioral Impairments Induced by Vandetanib of NeuN-positive cells and some co-localized with
Administration in 6-OHDA-Lesioned Rats caspase-3 (Fig. 5a, b). In fact, caspase-3 immunoreactivity
in the striatum and SN was more pronounced in the
The behavioral effect after vandetanib administration in vandetanib-treated rats (Fig. 5a, b), showing perinuclear
Parkinsonian rats was assessed by the amphetamine-induced localization.
rotational test. The results were expressed as the difference in
the number of rotations before and after treatment (increase or
Quantitative Analysis of the Effect Induced by Vandetanib
decrease).
Administration in 6-OHDA-Lesioned Rats
Two weeks after 6-OHDA lesion induction, no rats turned
more than 3 tpm (mean ipsilateral turns/min = 0.92 ± 0.2 tpm,
Vandetanib Increased the Loss of TH-Positive Terminals
n = 16). Therefore, this model was considered a prodromal or
in the Striatum After 6-OHDA Injection
preclinical model.
Three weeks after injury, the vandetanib-treated rats showed
The intrastriatal injection of 6-OHDA caused an increase in
the highest number of ipsilateral rotations. The number of rotations
the negative volume of the CPC in the TH-immunoreaction
was significantly greater than in the control group (Δ0.52 ± 0.19
(Fig. 2b), which was highest after vandetanib administration.
vs Δ0.08 ± 0.16 tpm, *P < 0.05; Student’s t test) (Fig. 1).
Therefore, in the vandetanib-treated group, the TH-negative
The rotations were recorded every 5 min for 90 min to
volume was 2.09 ± 0.13 mm3 (72.28 ± 3.64% of the total
show the differences in amphetamine metabolism to find
volume of the ipsilateral CPC), and it was 1.31 ± 0.2 mm3
the most active time prior to stabilization. The data were
in the control group (33.9 ± 58%) (***P < 0.001; Student’s t
also expressed as the increase or decrease in the number
test) (Fig. 2b). This difference was also evident when comparing
of rotations before and after treatment (Fig. 1). Vandetanib
the topological distributions. In the control group, the highest
administration did not change the time course of the am-
percentage of TH-negative volume was found in the middle level,
phetamine effects since the number of rotations began to
which corresponded to the levels located closer to the 6-OHDA
decline at 30–35 min and tended to stabilize in both
injection sites. Moreover, statistically significant differences were
groups after 50 min (Fig. 1).
found within the control group between the middle and caudal
sections (44.33 ± 1.75 and 21.52 ± 4.18%, respectively)
Morphological Changes Induced by Vandetanib
(*P < 0.05; one-way ANOVA). Remarkably, differences were
Administration in 6-OHDA-Lesioned Rats
also found between the caudal sections of both groups
(#P < 0.05; Student’s t test) (Fig. 2c). The vandetanib-treated
After the last behavioral assessment, the rats that were proc-
group showed the most abundant denervation (% of
essed for the morphological evaluation exhibited three marks
TH-negative volume) within the caudal levels of the striatum. In
on brain surfaces that corresponded to the sites of 6-OHDA
fact, significant differences were observed between the rostral
injection, but no macroscopic differences were observed be-
(57.61 ± 7.68%) and middle sections (66.71 ± 10.13%) compared
tween the groups.
to the caudal sections (84.95 ± 5.45%) within this group
Both groups showed a reduction in TH-positivity. The
(***P < 0.001 for rostral sections and *P < 0.05 for middle
control group showed more remarkable reductions in the
sections; one-way ANOVA) (Fig. 2c).
middle sections (Fig. 2a), and the vandetanib group
showed more noticeable reductions in the dorsolateral part
of striatum, with an obvious gradient from rostral to caudal Quantitative Evaluation of TH-Ir Neurons
sections (Fig. 2a). and the Axodendritic Network in the Substantia Nigra
Regarding the SN, the loss of positivity for TH was focused
on the external SN [17] (Fig. 3a). The rats that received van- TH-ir neurons were counted in the entire SN and in the external
detanib presented an outstanding loss of TH-positive neurons SN, and the TH-ir ADN was measured in the SNr and in the
and fibers (Fig. 3a). eSN. The values were expressed as the percent in the lesioned
Denervation and cell loss induced by 6-OHDA injections hemisphere compared to the contralateral hemisphere.
were accompanied by astrogliosis, which was shown by Regarding the entire SN, the control group showed a loss of
GFAP immunostaining. However, vandetanib administration dopaminergic neurons that was not modified by vandetanib
reduced GFAP positivity in the striatum and the SN in the treatment (control group, with only 29.72 ± 3.17%, vs the
lesioned hemisphere (Fig. 4a, c). vandetanib group with 26.53 ± 2.64% of TH-ir neurons)
Immunostaining for NeuN and caspase-3 revealed no (Fig. 3b). However, when the eSN was considered, the
differences in the control group when the lesioned vandetanib-treated group showed the lowest density of TH-ir
hemisphere was compared to the non-lesioned hemisphere, neurons (22.80 ± 4.33%) compared to the control group
but the vandetanib-treated group showed lower expression (39.41 ± 4.93%) (*P < 0.05; Student’s t test) (Fig. 3c).
Mol Neurobiol (2018) 55:201–212
Fig. 1 Behavioral effects after vandetanib administration. a The results
are expressed as the difference in the number of rotations before (week 0)
and after (week 1) treatment. The vandetanib group vs the control group
shows statistically significant differences (*P < 0.05). b The rotational
The TH-ir ADN was also reduced by 6-OHDA
intrastriatal injection, but the reduction was higher after
vandetanib treatment (Fig. 3d, e). The TH-ir ADN that
remained in the ipsilateral side from the eSN and from
Fig. 2 TH immunostaining in the striatum. a Photomicrographs of the
rostro-caudal distribution after 6-OHDA injection and vandetanib
administration. Scale bar = 2 mm. The arrow indicates the enlarged
ventricle in the lesioned hemisphere due to scar retraction after 6-
OHDA injection. b The graph shows the results obtained after
measuring the TH-negative volume. The results are expressed as the
percentage of TH-negative volume of the ipsilateral striatum relative to
205
behavior induced by an amphetamine was recorded every 5 min for
90 min; 50 min after administration, the effects of the amphetamine
diminished. None of the groups showed a difference in the metabolism
of the amphetamine
the SNr in the vandetanib-treated group significantly de-
creased compared to the control group (45.35 ± 5.54% in
the eSN and 30.08 ± 3.53% in the SNr vs 64.54 ± 8.51% in
the eSN and 59.23 ± 5.62% in the SNr). Statistically
the contralateral striatum. The vandetanib group showed the highest
percentage of TH-negativity relative to the control group (***P < 0.01).
c A graph that shows the percentage of TH-negative volume along the
rostro-caudal axis. An increasing rostro-caudal gradient was evident in
the vandetanib group, which was significantly different in the rostral and
caudal sections (*P < 0.05). In the control group, this gradient decreased
significantly from the middle to caudal sections (*P < 0.05)
206 Mol Neurobiol (2018) 55:201–212

Fig. 3 Vandetanib administration

induced neurodegeneration in the
substantia nigra (SN). a
Photomicrographs of the TH-
immunostained SN in every
group. Scale bar = 1 mm. b, c The
graphs show the density of
neurons in the whole SN and in
the external SN (eSN). The
vandetanib group showed the
lowest neuronal density in the
eSN (*P < 0.05). d, e The
vandetanib group showed a more
significantly decreased density of
the axodendritic network in the
SNr (***P < 0.001) than in the
external SN (**P < 0.01). The
results are expressed as the
percentage of the ipsilateral SN vs
the contralateral side

significant differences were observed between both groups
in both regions of the SN, but these differences were more
remarkable when the SNr was evaluated (**P < 0.01 in
eSN and ***P < 0.001 in SNr; Student’s t test) (Fig. 3d,
e). On the other hand, there were no differences along the
rostro-caudal axis within the groups in the SN.
GFAP Expression in the Striatum and the Substantia
Nigra
The quantification of GFAP immunoreactivity was devel-
oped by measuring the IOD in the striatum and the SN.
GFAP expression levels were not modified by 6-OHDA
intrastriatal injection (% in the lesioned side compared to
the non-lesioned hemisphere; 107.9 ± 3.15% in the
striatum and 100.6 ± 4.13% in the SN from the control
group) (Fig. 4). However, after vandetanib administration,
the GFAP immunoreactivity was lower in the lesioned side
compared to the non-lesioned hemisphere in the striatum
and in the SN (86.2 ± 3.83% in the striatum and
85.07 ± 4.19% in the SN) and compared with the lesioned
side from the control rats (Fig. 4b, d). Furthermore,
statistically significant differences between both groups
were more remarkable in the striatum than in the SN
(**P < 0.01 in the striatum and *P < 0.05 in the SN;
Student’s t test) (Fig. 4b, d).
Mol Neurobiol (2018) 55:201–212
Fig. 4 GFAP expression decreased after vandetanib administration. a, b
Photomicrographs of GFAP-immunostained striatum and substantia nigra
(SN) in every group. Scale bar = 50 μm. c A graph that shows the
percentage of the integrated optical density (IOD) in the striatum. The
Biochemical Changes Induced by Vandetanib
Administration in 6-OHDA-Lesioned Rats
The western blot analysis was carried out to evaluate the
changes in the apoptotic process (caspase-3 expression)
and the survival pathways (Akt and ERK signaling) in
the striatum and the SN in 6-OHDA-lesioned rats.
β-Actin was used as a loading control for caspase-3 and
for Akt levels, and β-tubulin was used as a loading control
for ERK 1/2 levels. Caspase-3 activation was expressed as
the percentage of the caspase-3/β-actin ratio of the le-
sioned hemisphere compared to the non-lesioned hemi-
sphere, set as 100%. p-Akt and p-ERK 1/2 levels were
normalized by total Akt and total ERK 1/2 levels, respec-
tively. The activations of Akt and ERK 1/2 were evaluated
as the percentage of p-Akt or p-ERK 1/2 relative to Akt or
ERK 1/2, respectively, in the ipsilateral hemisphere com-
pared to the contralateral hemisphere, set as 100%.
207
vandetanib group showed significantly decreased positivity for GFAP
(**P < 0.01). d In the SN, the positivity for GFAP also decreased in the
vandetanib group (*P < 0.05). The results are expressed as the percentage
of IOD in the lesioned hemisphere relative to the control hemisphere
Caspase-3 Activation by 6-OHDA Injection Was
Significantly Increased by Vandetanib
Caspase-3 activation was more remarkable in the SN
than in the striatum, probably due to the incipient loss
of dopaminergic neurons after 6-OHDA injection (Fig. 5).
Moreover, the vandetanib-treated group significantly
showed the highest caspase-3 levels in the striatum
(134.3 ± 14.19 vs 88 ± 8%) (*P < 0.05; Student’s t test)
a n d i n th e S N c om p a r e d t o t h e c o nt r ol g r o u p
(144.7 ± 0.9 vs 116.7 ± 2.73%) (*P < 0.05; Student’s t
test) (Fig. 5c, d).
Effects of Vandetanib on the Activation of Akt and Erk 1/2
in 6-OHDA-Lesioned Rats
Vandetanib administration decreased the levels of p-Akt/
Akt (99 ± 10.64% in the striatum and 81.67 ± 7.45% in
208
Fig. 5 Molecular changes in survival pathways after vandetanib administration. No differences were found in the activation of Akt (a) or ERK 1/2 (b) in
the vandetanib group relative to the control group
the SN) and p-ERK/ERK 1/2 (88.33 ± 7.69% in the
striatum and 83.33 ± 11.86% in the SN) in the ipsilateral
hemisphere compared to the contralateral hemisphere, in-
dicating that the inhibition of VEGFR2 and RET pro-
duced negative effects on cellular survival (Fig. 6).
Discussion
The present study demonstrated, for the first time, that
the inhibition of VEGFR2 and RET aggravated the
effects of 6-OHDA injection on the dopaminergic system
as shown by morpho-functional impairment, decreased
survival-pathway activity, and increased apoptotic-pathway
activity after vandetanib treatment. Therefore, these findings
could help to clarify whether the combination of VEGF
and GDNF represents a beneficial role in the restoration
of the nigrostriatal pathway and to support the synergis-
tic effect between NTFs as previously reported [16–19].
Mol Neurobiol (2018) 55:201–212
Morpho-functional Impairments After the Inhibition
of VEGFR2 and RET
Two weeks after 6-OHDA intrastriatal injection in adult rats,
only mild motor impairments were observed, but an incipient
dopaminergic loss was evident. After vandetanib administra-
tion, motor deficits and morphological changes were en-
hanced, showing a drastic loss of TH-positive volume in the
striatum accompanied by a remarkable loss of TH-ir neurons
and the ADN in the SN. The morphological analysis showed
that motor activity impairment was related to the degree of
denervation in the dorsal subregion of the CPC.
Unlike the noticeable loss of TH+ fibers in the striatum after
vandetanib administration, neuronal degeneration in the whole
SN was not remarkably different in the vandetanib-treated rats
compared to the rats that were only injected with the toxin.
However, consistent with our previous studies [17, 19], we
found significant results in the eSN, suggesting that this area
of the SN was more vulnerable to the effects of vandetanib
administration. Therefore, the inhibition of VEGF and GDNF
Mol Neurobiol (2018) 55:201–212
Fig. 6 Vandetanib administration
induced activation of caspase-3.
The vandetanib group showed
significantly increased expression
of caspase-3 relative to the control
group in the striatum and in the
substantia nigra (*P ≤ 0.05)
receptors could likely prevent the ability of VEGF and GDNF
to spread from non-lesioned areas to damaged regions to restore
function and provide additional neuroprotection [25].
In addition, the inhibition of VEGFR2 and RET signifi-
cantly reduced the density of the ADN in the SNr and in the
eSN. This reduction was more evident in the SNr, probably
because most axodendritic processes occur in this region. In
fact, the sprouting response depends on the extent of the lesion
and the GDNF capacity to induce regrowth and enhance the
neurite extension of dopaminergic neurons [26, 27].
Therefore, the inhibition of NTF activity appears to decrease
209
the ability of the surviving neurons to reinnervate the lesioned
SN.
Altogether, these results allow for the postulation that the
inhibition of VEGFR2 and RET negatively affects the
nigrostriatal system, resulting in the transition from the pre-
clinical PD stage to a partial model of PD. Since GDNF acti-
vates RET in the striatum to maintain the homeostasis be-
tween midbrain dopaminergic neurons and striatal neuronal
activity [28], the inhibition of RET by vandetanib may block
this function and lead to a loss of dopaminergic neurons and a
consequent loss of dopaminergic fibers.
210
The Tyrosine Kinase Inhibitor Induced a Deleterious
Effect in the Preclinical Model
Although apoptotic events have been reported in the 6-OHDA
model [29, 30], the inhibition of VEGFR2 and RET receptors
induced by vandetanib administration remarkably increased
the apoptotic effect induced by 6-OHDA. This finding is con-
sistent with a study conducted in breast and MTC cancer that
indicated that TKI treatment could be effective in increasing
apoptosis [31, 32].
On the other hand, Akt and ERK can be activated after the
binding of growth factors on many specific cell-surface recep-
tors [33, 34], consequently suppressing downstream survival
pathways such as MAPK/ERK and P13k/Akt. In normal con-
ditions, p-Akt is highly expressed in dopaminergic neurons,
but p-Akt levels are decreased in PD [35]. In this context,
some studies have revealed that some NTFs protect neuronal
cells from the apoptosis induced by MPP+ and 6-OHDA by
activating the Akt pathway [36, 37]. However, in the present
study, 6-OHDA lesions did not activate survival pathways
(Akt and ERK 1/2 signaling) in either the striatum or SN
(Fig. 6). This is consistent with Azcona and collaborators
who did not observe significant changes in p-Akt and
p-ERK 2 protein levels in 6-OHDA-lesioned rats [38]. In ad-
dition, the inhibition of VEGFR2 and RET led to dopaminer-
gic neuronal loss and dopaminergic denervation with a de-
crease in the activity of the survival pathways (Akt and
ERK1/2 pathways) in the striatum and SN. These findings
were consistent with other studies that supported the benefi-
cial effects of the upregulation of the ERK pathway after ac-
tivation by NTFs in experimental models of PD [39, 40].
Therefore, we suggested that VEGF and GDNF receptors
could exert their neuroprotective actions in PD via Akt and
ERK signaling cascades and suppress the expression of
caspase-3.
Astrogliosis Was Altered by Vandetanib Administration
In PD, marked astrogliosis in the SN has been reported [41].
Accordingly, denervation in the striatum has been suggested
to be accompanied by astrogliosis in the first stage of the
disease [42, 43]. Therefore, authors have suggested that the
suppression of the astrocyte response in PD poses a deleteri-
ous effect [44]. Some studies have revealed that astrocytes
could increase neuronal survival [43], suggesting that reactive
astrocytes contribute to the neuroplasticity/neuroregeneration
of dopaminergic neurons after an insult [45, 46]. The present
study showed that astrocyte-specific GFAP staining decreased
in the striatum and the SN in vandetanib-treated rats compared
to rats that were injected with only 6-OHDA. In fact, reactive
astrocytes have been shown to secrete neurotrophic factors,
including GDNF, that display trophic effects in dopaminergic
neurons [47, 48]. Additionally, exogenously administered
Mol Neurobiol (2018) 55:201–212
VEGF reportedly increased the number of reactive astrocytes
and GDNF levels after 6-OHDA lesion induction [49].
Therefore, the suppression of VEGF and GDNF activity could
lead to a loss of glial reactivity in response to an injury, such as
6-OHDA toxicity.
In conclusion, behavioral, morphological, and biochemical
results showed a morpho-functional decline in constitutive
compensatory protection against an aggression on the dopa-
minergic system after the inhibition of VEGF and GDNF sig-
naling. These findings were consistent with other studies,
supporting the assertion that VEGF and GDNF separately
promote the sprouting of neurites in the striatum and SN and
induce protection to increase the survival of dopaminergic
neurons in the SN [14, 40]. The negative effects from
VEGFR2 and RET inhibition after the administration of the
TKI vandetanib confirm the usefulness of VEGF and GDNF
as therapeutic targets in PD and support the potent and syner-
gistic effects exerted by NTFs. Therefore, the search for treat-
ments that stimulate or introduce ligands (VEGF and GDNF)
of these receptors could generate a strategy that allows for an
understanding of the molecular mechanism of PD.
Acknowledgments The authors appreciate the support from the
University of the Basque Country (UPV/EHU) (UFI 11/32), the Basque
Government (GIC IT 901/16 and 747-13, PPG 17/51), BMinisterio de
Ciencia e Innovación^ SAF 2016-77758-R (AEI/FEDER, UE), and
SGIker (UPV/EHU). C. Requejo appreciates the UPV/EHU for a fellow-
ship subvention.
References
1. Hirsch EC, Jenner P, Przedborski S (2013) Pathogenesis of
Parkinson’s disease. Mov Disord 28:24–30. doi:10.1002/mds.
25032
2. Ramaswamy S, Kordower JH (2009) Are growth factors the an-
swer? Parkinsonism Relat Disord 15(Suppl 3):S176–S180. doi:10.
1016/S1353-8020(09)70809-0
3. Yasuda T, Mochizuki H (2010) Use of growth factors for the treat-
ment of Parkinson’s disease. Expert Rev Neurother 10:915–924.
doi:10.1586/ern.10.55
4. Airaksinen MS, Saarma M (2002) The GDNF family: signalling,
biological functions and therapeutic value. Nat Rev Neurosci 3:
383–394. doi:10.1038/nrn812
5. Kramer ER, Aron L, Ramakers GMJ et al (2007) Absence of Ret
signaling in mice causes progressive and late degeneration of the
nigrostriatal system. PLoS Biol 5:e39. doi:10.1371/journal.pbio.
0050039
6. Drinkut A, Tillack K, Meka DP et al (2016) Ret is essential to
mediate GDNF’s neuroprotective and neuroregenerative effect in
a Parkinson disease mouse model. Cell Death Dis 7:e2359. doi:10.
1038/cddis.2016.263
7. Ferrara N (2009) Vascular endothelial growth factor. Arterioscler
Thromb Vasc Biol 29:789–791. doi:10.1161/ATVBAHA.108.
179663
8. Shibuya M (2011) Vascular endothelial growth factor (VEGF) and
its receptor (VEGFR) signaling in angiogenesis: a crucial target for
anti- and pro-angiogenic therapies. Genes Cancer 2:1097–1105.
doi:10.1177/1947601911423031
Mol Neurobiol (2018) 55:201–212
9. Musumeci F, Radi M, Brullo C, Schenone S (2012) Vascular endo-
thelial growth factor (VEGF) receptors: drugs and new inhibitors. J
Med Chem 55:10797–10822. doi:10.1021/jm301085w
10. Storkebaum E, Lambrechts D, Carmeliet P (2004) VEGF: once
regarded as a specific angiogenic factor, now implicated in neuro-
protection. BioEssays 26:943–954. doi:10.1002/bies.20092
11. Lafuente JV, Argandoña EG, Mitre B (2006) VEGFR-2 expression
in brain injury: Its distribution related to brain-blood barrier
markers. J Neural Transm 113:487–496. doi:10.1007/s00702-005-
0407-0
12. Wick A, Wick W, Waltenberger J et al (2002) Neuroprotection by
hypoxic preconditioning requires sequential activation of vascular
endothelial growth factor receptor and Akt. J Neurosci 22:6401–
6407
13. Kaya D, Gürsoy-Ozdemir Y, Yemisci M et al (2005) VEGF protects
brain against focal ischemia without increasing blood-brain perme-
ability when administered intracerebroventricularly. J Cereb Blood
Flow Metab 25:1111–1118. doi:10.1038/sj.jcbfm.9600109
14. Yasuhara T, Shingo T, Kobayashi K et al (2004) Neuroprotective
effects of vascular endothelial growth factor (VEGF) upon dopami-
nergic neurons in a rat model of Parkinson’s disease. Eur J Neurosci
19:1494–1504. doi:10.1111/j.1460-9568.2004.03254.x
15. Yasuhara T, Shingo T, Muraoka K et al (2005) Neurorescue effects
of VEGF on a rat model of Parkinson’s disease. Brain Res 1053:10–
18. doi:10.1016/j.brainres.2005.05.027
16. Herrán E, Ruiz-Ortega JÁ, Aristieta A et al (2013) In vivo admin-
istration of VEGF- and GDNF-releasing biodegradable polymeric
microspheres in a severe lesion model of Parkinson’s disease. Eur J
Pharm Biopharm 85:1183–1190. doi:10.1016/j.ejpb.2013.03.034
17. Requejo C, Ruiz-Ortega JA, Bengoetxea H et al (2015)
Topographical distribution of morphological changes in a partial
model of Parkinson’s disease-effects of nanoencapsulated neuro-
trophic factors administration. Mol Neurobiol. doi:10.1007/
s12035-015-9234-y
18. Herrán E, Requejo C, Ruiz-Ortega JA et al (2014) Increased
antiparkinson efficacy of the combined administration of VEGF-
and GDNF-loaded nanospheres in a partial lesion model of
Parkinson’s disease. Int J Nanomedicine 9:2677–2687. doi:10.
2147/IJN.S61940
19. Requejo C, Ruiz-Ortega JA, Bengoetxea H et al (2016)
Morphological changes in a severe model of Parkinson’s disease
and its suitability to test the therapeutic effects of microencapsulat-
ed neurotrophic factors. Mol Neurobiol 1–14. doi: 10.1007/
s12035-016-0244-1
20. Knight ZA, Lin H, Shokat KM (2010) Targeting the cancer kinome
through polypharmacology. Nat Rev Cancer 10:130–137. doi:10.
1038/nrc2787
21. Lorusso PM, Eder JP (2008) Therapeutic potential of novel
selective-spectrum kinase inhibitors in oncology. Expert Opin
Investig Drugs 17:1013–1028. doi:10.1517/13543784.17.7.1013
22. Huo Z, Yu S, Hong S et al (2016) A systematic review and meta-
analysis of the risk of diarrhea associated with vandetanib treatment
in carcinoma patients. Onco Targets Ther 9:3621–3631. doi:10.
2147/OTT.S96830
23. Paxinos G, Watson C (2013) The rat brain in stereotaxic coordinates
Hard Cover Edition. Academic Press
24. Argandoña EG, Bengoetxea H, Bulnes S et al (2012) Effect of
intracortical vascular endothelial growth factor infusion and block-
ade during the critical period in the rat visual cortex. Brain Res
1473:141–154. doi:10.1016/j.brainres.2012.07.008
25. Yue X, Hariri DJ, Caballero B et al (2014) Comparative study of the
neurotrophic effects elicited by VEGF-B and GDNF in preclinical
in vivo models of Parkinson’s disease. Neuroscience 258:385–400.
doi:10.1016/j.neuroscience.2013.11.038
26. Kirik D, Rosenblad C, Björklund A (2000) Preservation of a func-
tional nigrostriatal dopamine pathway by GDNF in the intrastriatal
211
6-OHDA lesion model depends on the site of administration of the
trophic factor. Eur J Neurosci 12:3871–3882
27. Nakajima K, Hida H, Shimano Y et al (2001) GDNF is a major
component of trophic activity in DA-depleted striatum for survival
and neurite extension of DAergic neurons. Brain Res 916:76–84.
doi:10.1016/S0006-8993(01)02866-9
28. Kramer ER, Liss B (2015) GDNF-Ret signaling in midbrain dopa-
minergic neurons and its implication for Parkinson disease. FEBS
Lett 589:3760–3772. doi:10.1016/j.febslet.2015.11.006
29. Stott SRW, Barker RA (2014) Time course of dopamine neuron loss
and glial response in the 6-OHDA striatal mouse model of
Parkinson’s disease. Eur J Neurosci 39:1042–1056. doi:10.1111/
ejn.12459
30. Hernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D
(2013) Activation of GSK-3β and caspase-3 occurs in nigral dopa-
mine neurons during the development of apoptosis activated by a
striatal injection of 6-hydroxydopamine. PLoS One 8:1–13. doi:10.
1371/journal.pone.0070951
31. Kunnimalaiyaan M, Ndiaye M, Chen H (2006) Apoptosis-mediated
medullary thyroid cancer growth suppression by the PI3K inhibitor
LY294002. Surgery 140:1009-14–1009-15. doi:10.1016/j.surg.
2006.06.040
32. Spanheimer PM, Cyr AR, Gillum MP et al (2014) Distinct path-
ways regulated by RET and estrogen receptor in luminal breast
cancer demonstrate the biological basis for combination therapy.
Ann Surg 259:793–799. doi:10.1097/SLA.0b013e3182a6f552
33. Nicole O, Ali C, Docagne F et al (2001) Neuroprotection mediated
by glial cell line-derived neurotrophic factor: involvement of a re-
duction of NMDA-induced calcium influx by the mitogen-activated
protein kinase pathway. J Neurosci 21
34. Karakaya S, Kipp M, Beyer C (2007) Oestrogen regulates the ex-
pression and function of dopamine transporters in astrocytes of the
nigrostriatal system. J Neuroendocrinol 19:682–690. doi:10.1111/j.
1365-2826.2007.01575.x
35. Timmons S, Coakley MF, Moloney AM, O’Neill C (2009) Akt
signal transduction dysfunction in Parkinson’s disease. Neurosci
Lett 467:30–35. doi:10.1016/j.neulet.2009.09.055
36. Cui W, Li W, Han R et al (2011) PI3-K/Akt and ERK pathways
activated by VEGF play opposite roles in MPP+-induced neuronal
apoptosis. Neurochem Int 59:945–953. doi:10.1016/j.neuint.2011.
07.005
37. Francardo V, Bez F, Wieloch T et al Pharmacological stimulation of
sigma-1 receptors has neurorestorative effects in experimental par-
kinsonism. doi: 10.1093/brain/awu107
38. Azkona G, Sagarduy A, Aristieta A et al (2014) Buspirone anti-
dyskinetic effect is correlated with temporal normalization of dys-
regulated striatal DRD1 signalling in L-DOPA-treated rats.
Neuropharmacology 79:726–737. doi:10.1016/j.neuropharm.
2013.11.024
39. Fuqua JL, Littrell OM, Lundblad M et al (2014) Dynamic changes
in dopamine neuron function after DNSP-11 treatment: effects
in vivo and increased ERK 1/2 phosphorylation in vitro. Peptides
54:1–8. doi:10.1016/j.peptides.2013.12.007
40. Lindgren N, Francardo V, Quintino L et al (2012) A model of
GDNF gene therapy in mice with 6-hydroxydopamine lesions: time
course of neurorestorative effects and ERK1/2 activation. J
Parkinsons Dis 2:333–348. doi:10.3233/JPD-012146
41. Paulus W, Jellinger K (1991) The neuropathologic basis of different
clinical subgroups of Parkinson’s disease. J Neuropathol Exp
Neurol 50:743–755
42. Morales I, Sanchez A, Rodriguez-Sabate C, Rodriguez M (2016)
The astrocytic response to the dopaminergic denervation of the
striatum. J Neurochem:81–95. doi:10.1111/jnc.13684
43. Rossi D (2015) Astrocyte physiopathology: at the crossroads of
intercellular networking, inflammation and cell death. Prog
Neurobiol 130:86–120. doi:10.1016/j.pneurobio.2015.04.003
212
44. Halliday GM, Stevens CH (2011) Glia: initiators and progressors of
pathology in Parkinson’s disease. Mov Disord 26:6–17. doi:10.
1002/mds.23455
45. Pellegrini E, Diotel N, Vaillant-Capitaine C et al (2016) Steroid
modulation of neurogenesis: focus on radial glial cells in zebrafish.
J Steroid Biochem Mol Biol 160:27–36. doi:10.1016/j.jsbmb.2015.
06.011
46. Episcopo FL, Tirolo C, Testa N et al (2013) Reactive astrocytes are
key players in nigrostriatal dopaminergic neurorepair in the MPTP
mouse model of Parkinson’s disease: focus on endogenous
neurorestoration. Curr Aging Sci 6:45–55
Mol Neurobiol (2018) 55:201–212
47. Ben Haim L, Carrillo-de Sauvage M-A, Ceyzériat K, Escartin C
(2015) Elusive roles for reactive astrocytes in neurodegenerative
diseases. Front Cell Neurosci 9:278. doi:10.3389/fncel.2015.00278
48. Chen P-S, Peng G-S, Li G et al (2006) Valproate protects dopami-
nergic neurons in midbrain neuron/glia cultures by stimulating the
release of neurotrophic factors from astrocytes. Mol Psychiatry 11:
1116–1125. doi:10.1038/sj.mp.4001893
49. Tian Y, Tang C-J, Wang J et al (2007) Favorable effects of VEGF
gene transfer on a rat model of Parkinson disease using adeno-
associated viral vectors. Neurosci Lett 421:239–244. doi:10.1016/
j.neulet.2007.05.033