Regulation of c-Ret, GFR␣1, and GFR␣2 in the
Substantia Nigra Pars Compacta in a Rat Model of
Parkinson’s Disease
Sònia Marco,1 Josep Saura,2 Esther Pérez-Navarro,1 Marı́a José Martı́,2
Eduard Tolosa,2 Jordi Alberch1
1
Departament de Biologia Cel䡠lular i Anatomia Patològica, Facultat de Medicina, Universitat de
Barcelona, IDIBAPS, Casanova 143, E-08036 Barcelona, Spain
2
Servei de Neurologia, Hospital Clı́nic, IDIBAPS, Universitat de Barcelona, Villarroel 170, E-08036
Barcelona, Spain
Received 31 January 2002; accepted 3 May 2002
ABSTRACT: Glial cell line-derived neurotrophic
factor (GDNF) family members have been proposed as
candidates for the treatment of Parkinson’s disease because they protect nigral dopaminergic neurons against
various types of insult. However, the efficiency of these
factors depends on the availability of their receptors
after damage. We evaluated the changes in the expression of c-Ret, GFR␣1, and GFR␣2 in the substantia
nigra pars compacta in a rat model of Parkinson’s disease by in situ hybridization. Intrastriatal injection of
6-hydroxydopamine (6-OHDA) transiently increased cRet and GFR␣1 mRNA levels in the substantia nigra
pars compacta at 1 day postlesion. At later time points, 3
and 6 days, the expression of c-Ret and GFR␣1 was
downregulated. GFR␣2 expression was differentially
regulated, as it decreased only 6 days after 6-OHDA
injection. Triple-labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed
that changes in the expression of c-Ret, GFR␣1, and
GFR␣2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that
nigral neurons differentially regulate the expression of
GDNF family receptors as a transient and compensatory
response to 6-OHDA lesion. © 2002 Wiley Periodicals, Inc. J
INTRODUCTION
Parkinson’s disease (Hornykiewicz and Kish, 1987).
To date, the available pharmacological treatments do
not stop the progression of this neurodegenerative
illness. Neurotrophic factors have emerged as candidates for a neuroprotective therapy, because of their
ability to regulate the survival of specific neuronal
populations in the central nervous system (Connor
and Dragunow, 1998). Several neurotrophic factors
prevent the degeneration of nigrostriatal dopaminergic neurons in different experimental models of Parkinson’s disease, such as 6-hydroxydopamine (6OHDA) injection (Levivier et al., 1995; Shults et al.,
2000; Spina et al., 1992), 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) treatment (Galpern et al.,
The degeneration of nigrostriatal dopaminergic neurons is the main neuropathological feature observed in
Correspondence to: J. Alberch (alberch@medicina.ub.es).
Contract grant sponsor: CICYT (Ministerio de Educación y
Ciencia, Spain); contract grant number: SAF99-0019.
Contract grant sponsor: Fundació La Marató de TV3; contract
grant number: 97-1009.
Contract grant sponsor: Fundació La Caixa; contract grant
number: 00/057-00.
Contract grant sponsor: Fundación Ramón Areces.
© 2002 Wiley Periodicals, Inc.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/neu.10082
Neurobiol 52: 343–351, 2002
Keywords: GDNF receptor family; 6-OHDA; in situ hybridization
343
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Marco et al.
1996; Spina et al., 1992; Tomac et al., 1995), and
medial forebrain bundle transection (Beck et al.,
1995; Lu and Hagg, 1997; Tseng et al., 1997). Among
them, glial cell line-derived neurotrophic factor
(GDNF) is one of the most potent neuroprotective
factors for these neurons (Akerud et al., 1999;
Lapchak et al., 1997; Sauer et al., 1995). Other members structurally related to GDNF, such as neurturin
(NRTN), artemin, and persephin, have recently been
described and shown to promote the survival and
activity of midbrain dopaminergic neurons (for review see Airaksinen et al., 1999).
GDNF family members exert their effects through
a two-component receptor complex that consists of a
common receptor protein kinase termed c-Ret (Jing et
al., 1996; Treanor et al., 1996; Trupp et al., 1996) and
one component of the GDNF family receptor-␣
(GFR␣) of glycosyl-phosphatidylinositol linked receptors (GFR␣1–GFR␣4), which behave as ligandbinding domains (Airaksinen et al., 1999; Baloh et al.,
2000). According to the trophic activity of GDNF
family members in the nigrostriatal system, c-Ret and
GFR␣s are expressed in the substantia nigra pars
compacta (SNpc) during development and throughout
adulthood (Glazner et al., 1998; Golden et al., 1999;
Horger et al., 1998; Masure et al., 2000; Nosrat et al.,
1997). Furthermore, GDNF and NRTN, the most representative members of the GDNF family, are expressed in the striatum (Golden et al., 1998; Naveilhan et al., 1998; Trupp et al., 1997; Widenfalk et al.,
1997), suggesting a target-derived trophic action on
nigrostriatal neurons (Akerud et al., 1999).
Endogenous neuroprotective mechanisms are activated by various types of brain injury. In this regard,
mRNA and protein levels of several trophic factors
are modified after the selective destruction of nigrostriatal dopaminergic neurons (Chadi et al., 1994;
Funa et al., 1996; Zhou et al., 1996). GDNF family
members are also regulated after lesion to dopaminergic neurons, but contradictory results have been reported. An increase in GDNF protein (Yurek and
Fletcher-Turner, 2001) and mRNA (Zhou et al., 2000)
levels in the striatum has been observed after
6-OHDA lesion. However, other authors have failed
to detect changes in striatal GDNF expression after
6-OHDA injection (Stromberg et al., 1993) or MPTP
treatment (Inoue et al., 1999). The neuroprotective
effects of GDNF and NRTN may depend not only on
the amount of these proteins but also on the availability of their receptors, c-Ret, GFR␣1, and GFR␣2.
Therefore, the understanding of the endogenous
mechanisms triggered by lesion to prevent neuronal
loss may facilitate the development of neuroprotective
strategies to avoid the degeneration of nigrostriatal
dopaminergic neurons.
The aim of the present study was to evaluate the
changes in the expression of c-Ret, GFR␣1, and
GFR␣2 in the SNpc induced by intrastriatal 6-OHDA
injection. Expression levels were analyzed by in situ
hybridization and the cellular localization of these
changes was characterized by triple labeling, consisting of in situ hybridization for GDNF receptors and
immunohistochemistry using specific neuronal and
glial cell markers.
METHODS
6-OHDA Lesions
Adult male Sprague-Dawley rats (Charles River, Saint
Aubin, France) weighing 300 – 400 g were anaesthetized
with sodium pentobarbital (50 mg/kg i.p.), after which they
received a single unilateral injection of 20 ␮g (4 ␮L) of
6-OHDA hydrochloride (Sigma, St. Louis, MO) or vehicle
solution into the left striatum. 6-OHDA was dissolved in
saline solution containing 0.2 mg/mL ascorbic acid and kept
on ice protected from light. The toxin was injected stereotaxically into the left striatum through a 30-gauge cannula
fitted to a Hamilton microsyringe at a rate of 0.5 ␮L/min.
The cannula was slowly withdrawn 2 min after the end of
infusion. Stereotaxic coordinates were taken from the Paxinos and Watson atlas and were AP 1.0 mm from bregma, L
3.0 mm, and DV ⫺4.5 mm below the dura with the incisor
bar set at ⫺3.3 mm. After surgery, rats were housed separately with access to food and water ad libitum, in a colony
room maintained at constant temperature (19 –22°C) and
humidity (40 –50%) on a 12:12 h light/dark cycle. All animal-related procedures were approved by Local Committee
(99/1; University of Barcelona) and Generalitat de Catalunya (1094/99) in accordance with the European Communities Council Directive (86/609/EU).
In Situ Hybridization
Animals were killed by decapitation 1, 3, or 6 days after
surgery. Brains were removed, frozen on dry ice, and stored
at ⫺20°C. Cryostat coronal sections (14 ␮m) through the
whole substantia nigra were serially collected on silanecoated slides. Consecutive sections were processed for in
situ hybridization as described elsewhere (Canals et al.,
2001). Briefly, after prehybridization, the sections were
hybridized at 55°C for 16 h with radioactive antisense
cRNA probes. The probes for c-Ret (Trupp et al., 1996),
GFR␣1 (Naveilhan et al., 1997), and GFR␣2 (Naveilhan et
al., 1998) were labeled by in vitro transcription using T3
(c-Ret), T7 (GFR␣2), or SP6 (GFR␣1) RNA polymerase
(Promega, Madison, WI) and [35S]-UTP (Amersham Intl.,
Little Chalfont, UK). For control experiments, sense cRNA
riboprobes were obtained using T3 (GFR␣2) or T7 (c-Ret
Regulation of GDNF Receptors in the SNpc
and GFR␣1) RNA polymerase. Sections were rinsed in
standard saline citrate (1⫻ SSC) and then washed in 0.5⫻
SSC, 50% formamide at 63°C for 30 min. They were then
treated with 40 ␮g/mL RNase A at 37°C for 30 min, washed
in SSC (2 ⫻ 15 min at 63°C, 1 ⫻ 15 min at room temperature), dehydrated, and air dried. Following dry film autoradiography exposure for 7–20 days, sections were dipped
in LM-1 emulsion (Amersham Pharmacia Biotech, Uppsala,
Sweden) for 2 months at 4°C, developed in D-19 (Eastman
Kodak, Rochester, NY), fixed, and lightly counterstained
with Cresyl violet. Some sections were processed for immunohistochemistry after the in situ hybridization, as described elsewhere (Canals et al., 2001). Briefly, after the last
wash in SSC, sections were coincubated with the primary
antibody against glial fibrillary acidic protein (GFAP;
1:500; Dako A/S Glostrup, Germany) and neuron-specific
nuclear protein (NeuN; 1:100; Chemicon, Temecula, CA)
overnight at 4°C. After washing in phosphate-buffered saline (PBS; pH 7.4), the sections were coincubated with the
corresponding secondary antibody at room temperature for 2 h
[antirabbit-FITC conjugated; 1:100 (Vector Laboratories, Burlingame, CA) and antimouse-Texas Red conjugated; 1:200
(Jackson ImmunoResearch, West Grove, PA)], washed in PBS
for 1 h, and dipped as described above. Triple-labeling analysis
was performed using a confocal microscope.
345
Image analysis was performed in a blind coded fashion in
six sections (separated by 140 ␮m) per animal, in three
animals per condition. Images were captured using a DP50
camera attached to an Olympus microscope. To estimate the
expression levels, the area (in ␮m2) covered by emulsion
autoradiography grains was measured using the AnalySIS
program (Soft Imaging System GmbH, Germany), because
the number of grains is linearly proportional to the mRNA
expression levels (Laprade and Soghomonian, 1997). The
expression of c-Ret was examined in the whole SNpc, while
GFR␣1 and GFR␣2 expression was analyzed in three fields
(0.033 mm2 each) from medial to lateral SNpc.
ptotic cells in the SNpc first appear 5 days after
6-OHDA injection (Marti et al., 1997). The levels of
expression of c-Ret, GFR␣1, and GFR␣2 in the SNpc
of sham animals did not differ significantly from the
levels in the contralateral side at any of the time points
studied [Figs. 1(A), 2(A), and 3(A)]. In these animals,
the grain density was the highest for c-Ret while
GFR␣2 showed the lowest one, suggesting different
levels of expression for the GDNF family receptors
[Figs. 1(B,D), 2(B,D), and 3(B)].
When 6-OHDA was intrastriatally injected, c-Ret
mRNA levels in the SNpc neurons were upregulated
1 day after lesion [198 ⫾ 23% of contralateral SNpc;
Fig. 1(A,C)]. However, the expression levels of c-Ret
were dramatically downregulated 3 days after
6-OHDA injection [54 ⫾ 6% of contralateral SNpc;
Fig. 1(A)], and this decrease was maintained 6 days
after lesion [55 ⫾ 10% of contralateral SNpc; Fig.
1(A,E)]. Triple-labeling studies showed that the in
situ hybridization signal colocalized with the neuronal
marker NeuN in sham-injected and lesioned animals
[Fig. 1(B–E)], showing that c-Ret expression after
lesion was regulated in nigral neurons.
GFR␣s were differentially regulated by 6-OHDA
lesion. Like c-Ret, GFR␣1 mRNA levels in the SNpc
were increased [149 ⫾ 12% of contralateral SNpc;
Fig. 2(A,C)] 1 day after 6-OHDA intrastriatal injection and fell below contralateral levels at 3 [72 ⫾ 8%
of contralateral SNpc; Fig. 2(A)] and 6 days postlesion [64 ⫾ 8% of contralateral SNpc; Fig. 2(A,E)]. In
contrast to c-Ret and GFR␣1, GFR␣2 expression was
unchanged 1 and 3 days after 6-OHDA lesion [Fig.
3(A)], but fell below control levels at 6 days postlesion [66 ⫾ 8% of contralateral SNpc; Fig. 3(A,C)].
GFR␣1 and GFR␣2 mRNAs were expressed in neurons, even after damage [Figs. 2(B–E) and 3(B–C)],
as revealed by triple-labeling studies.
Statistical Analysis
DISCUSSION
Quantitative Analysis
For each condition and probe studied, the mean ⫾S.E.M. of
the SNpc ipsilateral to 6-OHDA injection was normalized to
data of the contralateral hemisphere. Groups were compared
using an unpaired Student’s t test. The null hypothesis was
rejected at p ⬍ .05.
RESULTS
We examined the changes in the expression of GDNF
and NRTN receptors in the SNpc after vehicle or
6-OHDA injection using in situ hybridization. Lesioned and sham-injected animals were analyzed 1, 3,
and 6 days after surgery, because silver-stained apo-
We have characterized the regulation of c-Ret,
GFR␣1, and GFR␣2 expression in the SNpc by
6-OHDA intrastriatal injection. GFR␣1 and c-Ret
mRNAs showed a similar response, increasing 1 day
postlesion and decreasing 3 and 6 days after 6-OHDA
injection. GFR␣2 expression was differentially regulated by 6-OHDA, because it decreased only 6 days
after lesion. Triple-labeling studies indicate that cRet, GFR␣1, and GFR␣2 are expressed by neurons in
control and 6-OHDA-lesioned animals.
The differences in grain density for c-Ret, GFR␣1,
and GFR␣2 signal observed suggest that GDNF family receptors show different levels of expression in the
346
Marco et al.
Figure 1 Expression of c-Ret is regulated by 6-OHDA intrastriatal injection. Quantification of
c-Ret mRNA levels in SNpc of sham and 6-OHDA injected animals (A). Values are the mean
(⫾S.E.M.) of three animals per condition; *p ⬍ .05 and **p ⬍ .01 compared with contralateral side.
Photomicrographs show triple-labeling in the SNpc 1 [(B) and (C)] or 6 days [(D) and (E)] after
vehicle [(B) and (D)] or 6-OHDA injection [(C) and (E)]. Neu-N positive neurons are labeled in red,
GFAP-positive astrocytes are labeled in green, and white grains correspond to the c-Ret expression
signal, assessed by radioactive in situ hybridization. Scale bar: 20 ␮m.
SNpc. In agreement with our results, a strong signal
for c-Ret and GFR␣1 has been previously associated
with dopaminergic neurons of the SNpc (Horger et al.,
1998; Nosrat et al., 1997; Trupp et al., 1997; Widenfalk et al., 1997), whereas the majority of GFR␣2
expression is not located on dopaminergic neurons but
Regulation of GDNF Receptors in the SNpc
347
Figure 2 GFR␣1 mRNA levels in SNpc are modified by 6-OHDA lesion. Levels of GFR␣1
expression were measured in SNpc of sham and 6-OHDA injected animals (A). Values are the mean
(⫾S.E.M.) of three animals per condition; *p ⬍ .05 compared with contralateral side. Photomicrographs show triple-labeling in the SNpc after vehicle [(B) and (D)] or 6-OHDA injection [(C) and
(E)]. Red labeling corresponds to the neuronal marker Neu-N, green labeling represents GFAPpositive astrocytes, and white grains correspond to the GFR␣1 radioactive in situ hybridization
signal. Scale bar: 20 ␮m.
on cells residing nearby (Horger et al., 1998; Wang et
al., 2000; Widenfalk et al., 1997). After 6-OHDA
lesion, the mRNA levels of c-Ret, GFR␣1, and
GFR␣2 were modified, but the hybridization signal
remained in neurons, suggesting that astrocytic cells
are not involved in this endogenous trophic response.
348
Marco et al.
Figure 3 GFR␣2 expression is only modified 6 days after 6-OHDA injection. GFR␣2 mRNA
levels were quantified in SNpc at 1, 3, and 6 days after vehicle or 6-OHDA injection (A). Values
are the mean (⫾S.E.M.) of three animals per condition; *p ⬍ .05 compared with contralateral side.
Photomicrographs show triple-labeling in the SNpc after 6 days of vehicle (B) or 6-OHDA injection
(C). Neu-N positive neurons are labeled in red, GFAP-positive astrocytes are labeled in green, and
white grains correspond to the GFR␣2 expression, assessed by radioactive in situ hybridization.
Scale bar: 20 ␮m.
Our results show that c-Ret, GFR␣1, and GFR␣2
mRNA levels were differentially regulated shortly
after 6-OHDA injection. The expression levels of
GFR␣1 and c-Ret were upregulated, whereas GFR␣2
was not modified at 1 day postlesion. Therefore, lesion-induced changes in mRNA levels of GDNF receptors may modulate the response to endogenous
GDNF and NRTN. Although GDNF binds preferentially to GFR␣1 and NRTN to GFR␣2, there is some
cross-reactivity (Baloh et al., 1997; Buj-Bello et al.,
1997; Klein et al., 1997; Sanicola et al., 1997). In this
regard, it has been proposed that the protective effects
of NRTN on dopaminergic neurons are mediated by
GFR␣1 owing to the low expression of GFR␣2 in the
SNpc (Burazin and Gundlach, 1999; Horger et al.,
1998; Widenfalk et al., 1997). Furthermore, neither
GDNF nor NRTN exert any trophic effect on cultured
dopaminergic neurons obtained from GFR␣1 null mutant mice, and the addition of soluble GFR␣1 restores
the survival-promoting effects of both neurotrophic
factors (Cacalano et al., 1998; Wang et al., 2000).
These data point to the relevance of the GFR␣1/c-Ret
complex in the neurotrophic effects of GDNF and
NRTN on nigrostriatal dopaminergic neurons. Therefore, the increase in c-Ret and GFR␣1 expression
detected 1 day after intrastriatal 6-OHDA injection
may indicate a trophic response to increase the survival of dopaminergic neurons after lesion.
The transient upregulation of c-Ret and GFR␣1
mRNA levels was followed by a decrease in their
Regulation of GDNF Receptors in the SNpc
expression 3 and 6 days after 6-OHDA injection. This
downregulation (about 35%, present results) is similar
to the decrease in the number of tyrosine hydroxylasepositive cells that takes place 5 days after intrastriatal
6-OHDA injection (Marti et al., 2000). Therefore,
these changes in mRNA levels may be due to the
onset of dopaminergic neuron atrophy or death, because apoptotic profiles in the SNpc first appear at this
time point (Marti et al., 1997, 2000). Accordingly, 6
weeks after intrastriatal 6-OHDA injection, the loss of
dopaminergic cell bodies in the SNpc is accompanied
by reduced GFR␣1 expression (Sarabi et al., 2001)
and c-Ret immunoreactivity (Araujo et al., 1997). The
regulation of GFR␣2 expression was independent of
that of c-Ret and GFR␣1, as it was only reduced 6
days after lesion. This decrease could be related to the
atrophy of the small population of dopaminergic cells
that express GFR␣2 mRNA. However, we cannot rule
out that adjacent cells could be involved in the
changes observed for this receptor after 6-OHDA
injection.
Although GDNF is the most potent neuroprotective factor for dopaminergic neurons, other neurotrophic factors are also involved in the endogenous protective response activated by nigrostriatal lesion. For
instance, the expression of brain-derived neurotrophic
factor (BDNF) and its receptor TrkB is transiently
upregulated in the nigrostriatal system after axotomy
(Venero et al., 2000), while 6-OHDA injection increases BDNF protein levels in the striatum and in the
ventral midbrain (Yurek and Fletcher-Turner, 2001;
Zhou et al., 1996). Other trophic factors, such as basic
fibroblast growth factor (Chadi et al., 1994) or platelet-derived growth factor (Funa et al., 1996), are also
upregulated in the striatum and substantia nigra after
6-OHDA lesion. All these results show that compensatory mechanisms consisting of the regulation of the
expression of neurotrophic factors and/or their receptors are activated to avoid the lesion of nigrostriatal
dopaminergic neurons. Thus, the trophic support to
dopaminergic neurons is due to a complex interaction
between several neurotrophic factors and their receptors.
In conclusion, our results show that GDNF receptor family members are differentially regulated in
SNpc after intrastriatal 6-OHDA injection. c-Ret and
GFR␣1 expression in neurons of SNpc transiently
increases shortly after lesion, suggesting that the
GFR␣1/c-Ret complex is involved in the regulation of
dopaminergic neuron survival. Therefore, changes in
the availability of GDNF receptors after damage
should be taken into account to optimize neuroprotective treatments.
349
The authors thank Dr. Ernest Arenas, Dr. Patrick Ernfors, and Dr. Carlos Ibáñez from the Karolinska Institute
(Sweden) for the generous gift of the probes, Maite Muñoz
for technical assistance, and Anna Bosch and the Serveis
Cientı́fico-Tècnics (Universitat de Barcelona) for support
and advice in the use of confocal microscopy. S. M. was a
fellow of the CIRIT.
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