www.elsevier.com/locate/ynbdi

Neurobiology of Disease 23 (2006) 533 – 542

Selective injury to dopaminergic neurons up-regulates GDNF in

substantia nigra postnatal cell cultures: Role of neuron–glia crosstalk

Ana Saavedra,a Graca Baltazar,b Paulo Santos,c

Caetana M. Carvalho,a,d and Emılia P. Duartea,d,*

aCenter for Neuroscience and Cell Biology, University of Coimbra, PortugalbDepartment of Health Sciences, University of Beira Interior, PortugalcCenter for Histocompatibility, Coimbra, PortugaldDepartment of Zoology, University of Coimbra, Portugal

Received 15 February 2006; revised 27 March 2006; accepted 24 April 2006

Available online 12 June 2006

The effect of selective injury to dopaminergic neurons on the

expression of glial cell line-derived neurotrophic factor (GDNF) was

examined in substantia nigra cell cultures. H2O2, mimicking increased

oxidative stress, or L-DOPA, the main symptomatic treatment for

Parkinson’s disease, increased GDNF mRNA and protein levels in a

time-dependent mode in neuron–glia mixed cultures. The concentra-

tion dependence indicated that mild, but not extensive, injury induced

GDNF up-regulation. GDNF neutralization with an antibody decreased

dopaminergic cell viability in H2O2-treated cultures, showing that up-

regulation of GDNF was protecting dopaminergic neurons. Neither

H2O2 nor L-DOPA directly affected GDNF expression in astrocyte

cultures, but conditioned media from challenged mixed cultures

increased GDNF mRNA and protein levels in astrocyte cultures,

indicating that GDNF up-regulation was mediated by neuronal factors.

Since pretreatment with 6-OHDA completely abolished H2O2-induced

GDNF up-regulation, we propose that GDNF up-regulation is triggered

by failing dopaminergic neurons that signal astrocytes to increase

GDNF expression.

D 2006 Elsevier Inc. All rights reserved.

Keywords: Astrocytes; Crosstalk; Dopaminergic neurons; GDNF; Injury;

Neuroprotection; Parkinson’s disease; Substantia nigra

Introduction

Neurotrophic factors have attracted increasing attention be-

cause, in addition to promoting the survival and differentiation of

developing neurons, they protect neurons against injury (Connor

0969-9961/$ - see front matter D 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2006.04.008

* Corresponding author. Center for Neuroscience and Cell Biology,

Department of Zoology, University of Coimbra, 3004-517 Coimbra,

Portugal. Fax: +351 239 822776.

E-mail address: epduarte@cnc.cj.uc.pt (E.P. Duarte).

Available online on ScienceDirect (www.sciencedirect.com).

and Dragunow, 1998). Glial cell line-derived neurotrophic factor

(GDNF) was identified based on its ability to increase neurite

length, cell size, and the number of dopaminergic neurons as well

as their high-affinity dopamine uptake in culture (Lin et al., 1993).

GDNF was shown to be a potent factor for the protection of nigral

dopaminergic neurons against toxin-induced degeneration in

animal models of Parkinson’s disease (PD) (Hoffer et al., 1994;

Bowenkamp et al., 1995; Tomac et al., 1995; Gash et al., 1996;

Rosenblad et al., 1998; Akerud et al., 2001). Due to its ability to

rescue dopaminergic neurons after toxin-induced injury and to

promote recovery of the motor deficit (Kordower et al., 2000;

Grondin et al., 2002), GDNF represents a new potential therapeutic

tool for PD (Hurelbrink and Barker, 2001; Gill et al., 2003).

Deficient neurotrophic support has been implicated in neuro-

degeneration (Siegel and Chauhan, 2000), and lower levels of

GDNF were reported in the substantia nigra of PD patients by

Chauhan et al. (2001), but not by Mogi et al. (2001). Considerable

evidence suggests that nervous tissue reacts to injury by increasing

the expression of neurotrophic factors, including GDNF (Bar et al.,

1998; Satake et al., 2000; Miyazaki et al., 2001; Ikeda et al., 2002).

Lesion of the nigrostriatal pathway increases GDNF expression in

the striatum (Zhou et al., 2000; Nakajima et al., 2001; Yurek and

Fletcher-Turner, 2001), but the effect in the substantia nigra was

seldom addressed (Inoue et al., 1999; Yurek and Fletcher-Turner,

2001). However, the protection afforded by the local administra-

tion of GDNF (Kearns and Gash, 1995; Sauer et al., 1995; Winkler

et al., 1996; Kozlowski et al., 2000) and the presence of GDNF

family receptor a1 in the substantia nigra (Trupp et al., 1997;

Sarabi et al., 2001) suggest a role for the production of GDNF in

substantia nigra, in addition to the neurotrophic activity of target-

derived GDNF.

Despite years of clinical experience, the hypothesis that l-

DOPA therapy for PD may enhance neuronal damage, and thus

accelerate the progression of the disease, is still controversial. In

vitro, l-DOPA toxicity is well documented (Mena et al., 1992;

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542534

Gille et al., 2002; Blessing et al., 2003; Mytilineou et al., 2003),

but l-DOPA has not been convincingly shown to be toxic to

dopaminergic neurons in vivo (Perry et al., 1984; Lyras et al.,

2002). Clinical studies suggest that l-DOPA does not enhance the

rate of disease progression in PD patients (Markham and Diamond,

1986; Blin et al., 1988; Diamond and Markham, 1990; Uitti et al.,

1993) and actually may slow the progression of familial PD

(Gwinn-Hardy et al., 1999). Studies in animal models of PD also

indicate that l-DOPA is not toxic and may even have trophic

effects (Melamed et al., 1985; Murer et al., 1998; Datla et al.,

2001). However, the effect of l-DOPA on GDNF levels in the

nigrostriatal system has never been addressed.

The present study aimed at investigating the effect of the

selective injury to dopaminergic neurons on GDNF expression in

the substantia nigra. Increased oxidative stress has been implicated

in the death of dopaminergic neurons, even in the genetic forms of

PD (Lotharius and Brundin, 2002). Dopamine breakdown cata-

lyzed by MAO generates H2O2, which is only slightly toxic by

itself but, in the presence of iron, can be broken down into the

more reactive hydroxyl radicals that rapidly react with DNA,

membrane lipids and proteins (Blum et al., 2001). In the early

stages of PD, the overactivity of the surviving dopaminergic

neurons may increase H2O2 production (Zigmond et al., 2002).

Moreover, at normal pH, dopamine can auto-oxidize into toxic

dopamine-quinones, superoxide radicals and H2O2. The oversup-

ply of dopamine due to l-DOPA therapy may also increase

oxidative stress. These oxidative stress-promoting reactions are

likely to occur when dopamine sequestration in vesicles is

compromised (Lotharius and Brundin, 2002). In addition, we have

shown that H2O2 and l-DOPA induce oxidative stress in our

model, as indicated by the up-regulation of heme-oxygenase-1

(Saavedra et al., 2005).

In the present work, we used rat substantia nigra postnatal cell

cultures to examine the effect of H2O2 and l-DOPA on GDNF

expression. The advantage of using postnatal cells cultures, instead

of embryonic cultures, is the more advanced development of

substantia nigra and ventral tegmental area (VTA) allowing the

more precise dissection of these two structures. This is particularly

important due to the differential sensitivity of dopaminergic

neurons from the VTA and the substantia nigra to toxic insults

(Ding et al., 2004). In addition, this postnatal cell culture is

appropriate to investigate the interplay between neuronal and glial

cells. We found that mild toxic damage to dopaminergic neurons

triggers GDNF up-regulation, which involves soluble mediators

that signal astrocytes to increase the expression of this neurotrophic

factor. Since the increase in GDNF expression was not observed in

6-hydroxydopamine (6-OHDA)-treated cultures, we propose that

challenged dopaminergic neurons can trigger GDNF up-regulation

as a neuroprotective strategy.

Materials and methods

Cell culture

Animals were handled in accordance with the national ethical

requirements for animal research and with the European Conven-

tion for the Protection of Vertebrate Animals Used for Experi-

mental and Other Scientific Purposes. Postnatal substantia nigra

neuron–glia cocultures were prepared as previously described by

Burke et al. (1998) with some modifications. To prepare the

cultures, a coronal slice at the level of midbrain flexure was

dissected from postnatal day 1–3 Wistar rat pups, followed by the

removal of the dorsal midbrain. The entire ventral segment of the

midbrain, including both the substantia nigra and the VTA, was

used to prepare astrocyte cultures. For the neuronal preparations, a

paramedian vertical cut was made left and right to separate left and

right substantia nigra from the central VTA (Smeyne and Smeyne,

2002). The tissue was then enzymatically dissociated under

continuous oxygenation using 20 U/ml of papain (Roche) in 1.0

mM cysteine, 116 mM NaCl, 5.4 mM KCl, 26 mM NaHCO3, 2

mM NaH2PO4, 1 mM MgSO4, 500 AM EDTA, 25 mM glucose

and 0.001% phenol red, at pH 7.3, for about 30 min at 33-C. Tostop digestion, tissue chunks were washed with culture medium.

The tissue was then mechanically dissociated, and the cell

suspension pelleted, resuspended and plated onto poly-d-lysine

and laminin-coated coverslips under 0.8 cm2 holes in the bottom of

50 mm snap-top polystyrene Petri dishes (BD Falcon). For

midbrain astrocyte cultures, 75 000 cells were plated and fed with

astrocyte culture medium, M10C-G (composition described by

Burke et al., 1998). Once the cells were confluent, 25 AM 5-

fluorodeoxyuridine with 70 AM uridine (FDU) was added to the

culture medium to suppress cell growth. The neuronal cultures

were established by plating 80,000 cells onto confluent monolayers

of midbrain astrocytes. Three days before the neuronal cell

preparation, the medium was changed to neuronal culture medium,

SF1C (composition described by Burke et al., 1998), to allow the

conditioning by astrocytes. Proliferation of non-neuronal cells was

suppressed by addition of FDU 1 day after plating. The cultures

were kept at 37-C in a 5% CO2, 95% air atmosphere. Substantia

nigra neuron–glia mixed cell cultures were used after 1 week in

culture, and astrocyte cultures were used after confluence was

reached. The day prior to cell treatment, the culture medium of

either mixed cell cultures or astrocyte cultures was replaced by

serum-free SF1C.

MTT assessment of cell viability

After the treatment, cultures were incubated with 3-(4,5-

dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)

(0.5 mg/ml) in Krebs medium for 60–90 min at 37-C. MTT is

converted by viable cells to a water-insoluble precipitate that was

dissolved in 0.04 M HCl in isopropanol, and colorimetrically

quantified (O.D. 570 nm) using a microplate reader.

Immunocytochemistry

After rinsing, the cells were prefixed in Minimal Essential

Medium with few drops of 4% paraformaldehyde, and fixed in 4%

paraformaldehyde for 10 min. The cells were then permeabilized

with 0.2% Triton X-100 in phosphate buffered saline (PBS) for 10

min, and blocked by incubation with 0.2% gelatin in PBS with

0.5% Tween-20 for 90 min at room temperature. The cells were

incubated with a mouse monoclonal anti-tyrosine hydroxylase

(TH) (Calbiochem; 1:10000) and a rabbit polyclonal anti-micro-

tubule associated protein 2 (MAP2) (Chemicon; 1:1000) antibodies

for 90 min at room temperature. After washing, the cells were

incubated with a goat anti-mouse IgG antibody conjugated to

Alexa FluorR 488 and a goat anti-rabbit IgG antibody conjugated

to Alexa FluorR 594 (Molecular Probes; 1 Ag/ml and 2 Ag/ml,

respectively). To assess the specificity of TH and MAP2

immunostaining, the primary antibody was omitted in some

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542 535

coverslips. No fluorescence was detected in these conditions. For

quantification purposes, fifteen randomly assigned fields, for each

condition, were analyzed on a Zeiss inverted microscope under a

200� magnification, and the cells immunoreactive to TH and

MAP2 counted. In each independent experiment, at least 250

MAP2-positive cells were counted for each condition. The

population of dopaminergic neurons was reported as the percentage

of the total neuronal population labeled by MAP2 antibody.

Western blot

Cells were lysed in buffer containing 25 mM Tris, 2.5 mM

EDTA, 2.5 mM EGTA, 1% Triton X-100, 1 mM dithiothreitol,

1 mM phenylmethylsulfonyl fluoride and 25 Ag/ml leupeptin.

Protein concentration was determined using Bradford’s method

with bovine serum albumin as standard. Equal amounts of protein

(usually 15–20 Ag) were separated by SDS–PAGE using a 12%

resolving gel under reducing conditions, and electrotransferred

onto polyvinylidene fluoride membranes (Amersham Life Scien-

ces). After being blocked with 5% milk powder in TBS-T (0.5%

Tween 20 in a 20 mM Tris and 137 mM NaCl solution), for 60 min

at room temperature, the membranes were incubated overnight at 4

-C with GDNF primary antibody, diluted 1:1000 (Santa Cruz

Biotechnology) in TBS-T containing 1% milk powder. After being

rinsed, blots were incubated for 60 min, at room temperature, with

an alkaline phosphatase-conjugated anti-rabbit antibody (Amer-

sham Life Sciences), diluted 1:20000 in TBS-T containing 1% milk

powder. Protein bands were detected using the Enhanced Chemi-

Fluorescence (ECF) system (Amersham Life Sciences) and

quantified by densitometric analysis using the Quantity One

software (Bio-Rad).

Total RNA extraction and reverse transcription

Total RNA was extracted from cell cultures using the TRIzolRreagent according to the manufacturer’s protocol (Invitrogen). The

isolated RNA was dissolved in 20 Al diethylpyrocarbonate-treatedwater and stored at �80-C. To remove any residual genomic DNA,

RNA samples were treated with DNase I (Invitrogen) for 15 min at

room temperature, which was then inactivated by incubation at

65-C for 10 min. To assess RNA integrity, randomly chosen

samples were analyzed in an Agilent 2100 Bioanalyzer using a

RNA NanoLabChipR (RNA 6000 Nano Assay, Agilent Tecnhol-

ogies). The RNA integrity number was greater than 7 in all

analyzed samples.

Single-stranded cDNAs were synthesized using TaqManRReverse Transcription Reagents (Roche Molecular Systems) by

incubating total RNA (0.5 Ag of DNase-treated RNA) for 10 min at

25-C, and 30 min at 48-C, in a final volume of 25 Al. The reactionwas terminated by incubating 5 min at 95-C. All samples were

stored at �20-C until analysis. For control purposes, non-template

samples were subjected to reverse transcription.

Real-time PCR

Real-time PCR was performed to monitor the expression of

GDNF and of a housekeeping gene, the 18S ribosomal RNA

(TaqMan ribosomal RNA Control Reagents) using the TaqMan

technology, and the results analyzed with a 7900 HT Sequence

Detector System (Applied Biosystems). Primers and the TaqMan

probe were selected from Genebank (accession number L15305)

and designed using the Primer Express software (Applied

Biosystems): forward primer-5V GACTTGGGTTTGGGCTAC-

GAA 3V; reverse primer-5V ATTGTCTCGGCCGCTTCAC 3V;TaqManR probe-5V 6-FAM AAGGAGGAACTGATCTTTCGA-

TATTGTAGCGGTTC 3VTAMRA. Specificity of the primers was

confirmed by a BLAST search. The amplification reaction mixture

(25 Al) contained the TaqManR Universal PCR Master Mix

components and 5 Al of the cDNA sample, 300 nM of each primer,

and 250 nM TaqManR probe (Applied Biosystems). The thermal

cycling conditions consisted of 10 min at 95-C, proceeding with 40cycles of 95-C for 15 s, and 60-C for 1 min. The size of the PCR

product was determined in an Agilent 2100 Bioanalyzer using a

DNA 1000 LabChipR Kit (Agilent Tecnhologies). The GDNF

mRNA levels were normalized to that of the 18S ribosomal RNA

and expressed relative to control using the DDCt method.

GDNF neutralization using an antibody

Substantia nigra cell cultures were incubated for 24 h with 100

AM H2O2 in the presence or absence of the anti-GDNF antibody

(Santa Cruz Biotechnology, 0.4 Ag/ml). Approximately 10 h later,

the application of the antibody was repeated to ensure that only

residual GDNF would be available to bind to its receptor. The

cultures were then processed for TH and MAP2 immunocyto-

chemistry as described above.

Exposure of neuron–glia mixed cultures to 6-OHDA

After 4 days in culture, the culture medium was withdrawn and

cells were exposed to 40–500 AM 6-OHDA in the presence of

1 mM of the metal chelator diethylenetriaminepentaacetic acid

(DETAPAC) in Minimal Essential Medium (Ding et al., 2004).

Control cultures were incubated with vehicle alone. After 15 min at

37-C, the incubation medium was removed and the cultures were

gently washed twice with warm Minimal Essential Medium before

adding the culture medium. Cultures were then kept at 37-C in a

5% CO2, 95% air atmosphere for further 3 days before the

experiments.

Results

Effects of H2O2 and l-DOPA on cell viability in substantia nigra

neuron–glia mixed cultures

To setup conditions for the selective damage of dopaminergic

neurons, we investigated the effects of H2O2 and l-DOPA

treatments on the viability of the overall cell population using

the MTT assay, and on the viability of dopaminergic cells by TH

immunocytochemistry. H2O2 (50–150 AM) or l-DOPA (50–400

AM) were added added to substantia nigra neuron–glia mixed cell

cultures, and cell viability was examined 24 h later. We did not

observe any statistically significant effect of H2O2 or l-DOPA on

the viability of the overall cell population (Fig. 1A), but we did

observe effects on dopaminergic cell viability (Fig. 1B). The

percentage of TH-positive cells decreased from 6.7 T 0.5% of the

total neuronal population in control cultures to 5.2 T 0.9% (about

20% dopaminergic cell loss), and to 4.1 T 0.3% (about 40%

dopaminergic cell loss) in cultures treated with 200 AM and 400

AM l-DOPA, respectively. No effect on dopaminergic cell viability

was observed with H2O2 in concentrations up to 150 AM. The

Fig. 1. Effect of H2O2 and l-DOPA on the viability of the overall cell population (A) and of dopaminergic neurons (B) in substantia nigra neuron–glia mixed

cultures. The cultures were incubated with H2O2 or l-DOPA for 24 h, and assayed for MTT reduction (A) or processed for TH and MAP2 immunoreactivity

(B). In each independent experiment, cell counting was performed in fifteen fields (at least 250 MAP2-positive cells were counted) for each condition. The

density of dopaminergic neurons (TH-positive, TH+) was evaluated as the percentage of MAP2 immunoreactive cells. The values are the percentage of

dopaminergic neurons as compared to control (Ctr). In control cultures 6.7 T 0.5% neurons were dopaminergic. Data are shown as the mean T SEM of at least

three independent experiments. Statistical analysis was performed using ANOVA followed by Dunnett’s test. **P < 0.01 and ***P < 0.001 as compared to

control.

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542536

percentage of dopaminergic neurons in control cultures (about 7%

of the total neuronal population) was consistent with data reported

previously by Burke et al. (1998) in substantia nigra postnatal cells

cultured without added GDNF on a feeding layer of cortical

astrocytes. We used ventral midbrain astrocytes since the origin of

the astrocyte layer was shown to influence the survival of

dopaminergic neurons in culture (Langeveld et al., 1995; Du et

al., 2005). Furthermore, astrocytes from distinct brain regions

appear to secrete unique combinations of factors important to

induce specific neuronal phenotypes. Only ventral midbrain

astrocytes were capable of inducing a dopaminergic phenotype in

a multipotent neural stem cell line (Wagner et al., 1999).

H2O2 and l-DOPA up-regulated GDNF in substantia nigra

neuron–glia cultures, but not in astrocyte cultures

To examine GDNF expression in conditions of dopaminergic

challenge, substantia nigra mixed cultures were treated for 24 h

with increasing concentrations of H2O2 or l-DOPA, and protein

Fig. 2. Effect of H2O2 and l-DOPA on GDNF levels in substantia nigra neuron–

incubated with the indicated concentrations of H2O2 or l-DOPA, and astrocyte cul

Protein extracts were prepared and separated by SDS-PAGE followed by immuno

blots for GDNF cell content. (B, D) Quantification of the Western blot data by den

(Ctr) incubated in the absence of the toxic stimuli. Data shown are the mean T SEM

analysis was performed using ANOVA followed by Dunnett’s test. *P < 0.05 an

extracts analysed by Western blot. Bell-shaped concentration–

response relationships were observed for the effects of H2O2 and l-

DOPA on GDNF levels (Figs. 2A and B). Only 100 AM H2O2, and

200 or 300 AM l-DOPA induced significant increases in GDNF

cell content, respectively 60 T 15.4%, 70 T 9.4% and 42.8 T 33.0%

above control. Further increasing the concentration of the toxic

stimuli did not change GDNF protein levels as compared to

controls. To check direct effects of H2O2 or l-DOPA on GDNF

expression by astrocytes, we investigated the effect of 24 h

incubation with 100 AM H2O2 or 200 AM l-DOPA on GDNF

protein levels in midbrain astrocyte cultures. No statistically

significant effect was observed (Figs. 2C and D). The time-course

of GDNF protein accumulation in mixed cultures treated with 100

AM H2O2 or 200 AM l-DOPA showed that GDNF protein levels

were significantly increased relatively to control only at 24 h

(Fig. 3).

Changes in GDNF expression were also examined by deter-

mining mRNA levels by real-time PCR analysis in substantia nigra

mixed cultures. Cell cultures treated with 100 AM H2O2 showed a

glia cultures (A, B) and in astrocyte cultures (C, D). Mixed cultures were

tures were incubated with 100 AM H2O2 or 200 AM l-DOPA, both for 24 h.

blot analysis using an anti-GDNF antibody. (A, C) Representative Western

sitometric analysis. The results were expressed as the percentage of controls

of at least three independent experiments carried out in triplicate. Statistical

d **P < 0.01 as compared to control.

Fig. 3. Time-course of H2O2 and l-DOPA effects on GDNF levels in

substantia nigra mixed cultures. The cultures were incubated with 100 AMH2O2 or 200 AM l-DOPA for the indicated periods of time. Protein extracts

were prepared and analyzed by Western blotting using an anti-GDNF

antibody. Quantification of the Western blot data for GDNF was performed

by densitometric analysis. The results were expressed as the percentage of

controls incubated in the absence of the toxic stimuli. Data are shown as the

mean T SEM of at least three independent experiments performed in

triplicate. Statistical analysis was performed using ANOVA followed by

Dunnett’s test. **P < 0.01 as compared to control.

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542 537

marked increase in GDNF mRNA at 1 h (3.65 T 1.00 fold the

control levels), decreasing to basal levels thereafter (Fig. 4). On

the other hand, in cultures incubated with 200 AM l-DOPA, the

increase in GDNF mRNA levels at 1 h was less pronounced

(1.96 T 0.26 fold), but more sustained over time (at 3 h, 2.19 T0.10 fold the control levels), returning to control levels at 6 h.

GDNF up-regulation protected dopaminergic neurons

To investigate whether the increase in endogenous GDNF was

protecting dopaminergic cells from H2O2 toxicity, we used an

antibody neutralization approach to prevent GDNF from reaching

its targets. GDNF neutralization significantly decreased the

percentage of TH-positive cells from 7.1 T 1.4% in mixed cultures

incubated with 100 AM H2O2 to 5.5 T 1.0% in cultures submitted

to the same treatment in the presence of the GDNF antibody. This

represents a dopaminergic cell loss of about 23% when GDNF was

Fig. 4. Effect of H2O2 and l-DOPA on GDNF mRNA levels in substantia

nigra mixed cultures. Cells were incubated with 100 AM H2O2 or 200 AMl-DOPA for up to 24 h, and total RNA extracted. For each sample, 0.5 Agof RNAwas reverse transcribed and analyzed by real-time PCR for GDNF

and for a housekeeping gene, the 18S ribosomal RNA, to normalize the

results. The expression levels from three to six independent experiments are

presented as mean T SEM relative to controls incubated in the absence of

stimuli. Statistical analysis was performed using ANOVA followed by

Dunnett’s test. **P < 0.01 as compared to control.

neutralized, whereas no effect was observed in the control (Fig. 5).

Therefore, GDNF neutralization uncovered the effect of H2O2 on

dopaminergic cell viability, and showed that endogenous up-

regulation of GDNF can protect dopaminergic neurons from H2O2-

induced toxicity.

Conditioned media from challenged substantia nigra neuron–glia

cultures increased GDNF expression in astrocyte cultures

Since we observed GDNF up-regulation in response to H2O2 or

l-DOPA in neuron–glia mixed cultures, but no effect in astrocyte

cultures (Fig. 2), we set out to investigate the role of neuronal cells in

the expression of GDNF. We determined the effect of media

conditioned for 24 h in substantia nigra mixed cell cultures treated

with 100 AM H2O2 or 200 AM l-DOPA, on GDNF levels in

midbrain astrocyte cultures. Small, but consistent, increases in

GDNF cell content to 132.6 T 7.7% and 119.4 T 4.0% of control were

observed upon incubation for 24 h with conditioned media from

H2O2- or l-DOPA-treated mixed cultures, respectively (Fig. 6A).

The up-regulation of GDNF observed in astrocyte cultures

incubated with conditioned media (20–30%) was significantly

smaller than the up-regulation observed in mixed cultures (60–

70% above control). To investigate whether the relatively small

effect of conditioned media could be due to degradation/

inactivation of the putative mediators during the 24-h period, we

tested the effects of media conditioned for shorter periods of time

on GDNF mRNA levels in astrocyte cultures. Substantia nigra

mixed cultures were incubated with 100 AM H2O2 or 200 AM l-

DOPA for 1, 3 or 6 h, and the conditioned media transferred to

astrocyte cultures for 1 h. We observed a 2.15 T 0.53 fold increase

in GDNF mRNA levels in astrocyte cultures exposed to medium

conditioned for 1 h in l-DOPA-treated cultures (Fig. 6B). The

conditioned media from H2O2-exposed cultures induced a trend to

increase GDNF mRNA levels in astrocyte recipient cultures, which

however did not reach significance. No effect was observed with

media conditioned for longer periods of time (3 or 6 h), as

compared to astrocytes incubated with conditioned medium from

control cultures (Fig. 6B). For control purposes, some astrocyte

cultures were incubated for 1 h with fresh serum-free medium.

These cultures exhibited 2.5-fold smaller GDNF mRNA levels

Fig. 5. Effect of GDNF neutralization on dopaminergic cell viability.

Substantia nigra mixed cultures were incubated for 24 h with H2O2 in the

presence (+) or in the absence (�) of the anti-GDNF antibody (0.4 Ag/ml;

with reinforcement approximately 10 h later) and processed for immuno-

cytochemistry. In each independent experiment, cell counting was

performed in fifteen fields (at least 250 MAP2-positive cells were counted)

for each condition. The values are the percentage of dopaminergic neurons

as compared to control, evaluated in relation to the total MAP2

immunoreactive neurons. *P < 0.05 as compared to the same condition

in the absence of the neutralizing antibody (Bonferroni’s test).

Fig. 6. Effect of conditioned media from substantia nigra mixed cultures on GDNF protein (A) and mRNA (B) levels in astrocyte cultures. Mixed cultures were

incubated in the absence (Ctr; time point 0 h) or in the presence of 100 AM H2O2 or 200 AM l-DOPA for 24 h (A) or for the indicated periods of time (B), and

the conditioned media transferred to astrocyte cultures for 24 h (A) or 1 h (B). (A) Protein extracts were analyzed by Western blot using an anti-GDNF

antibody. A representative Western blot for GDNF in astrocyte cultures upon treatment with conditioned media from mixed cultures is presented. Quantification

of the Western blot data was performed by densitometric analysis. Data are shown as the mean T SEM of up to five independent experiments made in triplicate.

(B) Total RNA extracts from astrocyte cultures incubated with conditioned media were prepared, reverse-transcribed and analyzed by real-time PCR to

determine GDNF mRNA levels relative to control cultures, incubated with control conditioned medium. Statistical analysis was performed using ANOVA

followed by Dunnett’s test. *P < 0.05 and **P < 0.01 as compared to control.

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542538

than astrocyte cultures incubated with conditioned medium from

control neuron–glia cultures (data not shown).

Pretreatment with 6-OHDA abolished H2O2-induced GDNF

up-regulation in substantia nigra mixed cultures

To examine the role of dopaminergic neurons in toxic stimuli-

induced GDNF up-regulation, we used the neurotoxin 6-OHDA to

selectively kill dopaminergic neurons in substantia nigra cell

cultures. 6-OHDA is selectively taken up by catecholaminergic

neurons and therefore is widely used both in vivo, to produce

animal models of PD, and in cell culture models. It undergoes a

rapid and non-enzymatic oxidation leading to the production of

reactive oxygen species (Soto-Otero et al., 2000). To find a

Fig. 7. Effect of 6-OHDA treatment on dopaminergic and non-dopaminergic neuro

induced by H2O2 (B). After 4 days in culture the cells were treated with 500 AM 6-

TH and MAP2 immunoreactivity (A) or challenged with H2O2 (B). Protein e

representative Western blot for GDNF cell content and the quantification of the We

mean T SEM of independent experiments performed in triplicate *P < 0.05, ***P <

(Bonferroni’s test). Scale bar: 20 Am.

concentration of 6-OHDA that selectively damaged dopaminergic

neurons while sparing the other cell types, we treated 4 days old

neuron–glia cultures with 6-OHDA (40–500 AM) for 15 or 30

min, and the effects on cell survival were examined 3 days later,

according to the protocol described by Ding et al. (2004) also using

a postnatal substantia nigra cell culture. The impact of the 6-

OHDA treatment on cell cultures was assessed by immunocyto-

chemistry for TH and MAP2. Unlike Ding et al. (2004), we did not

observe any sign of decreased dopaminergic cell viability with 6-

OHDA in the range 40–100 AM. Even upon the treatment with

500 AM 6-OHDA (15 min, followed by 3 days in culture), we still

observed TH-positive neurons in the culture but exhibiting much

shorter processes when compared to the highly branched dopami-

nergic neurons in control cultures (Fig. 7A). Non-dopaminergic

ns in substantia nigra neuron–glia cultures (A) and on GDNF up-regulation

OHDA(+) or vehicle (�) for 15 min at 37 -C, and 3 days later processed forxtracts were analyzed by Western blot using an anti-GDNF antibody. A

stern blot data by densitometric analysis are presented. Data are shown as the

0.001 as compared to control and ###P < 0.001 as compared to H2O2 alone

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542 539

neurons were not affected by the 6-OHDA incubation, as assessed

by MAP2 immunolabeling. Although we could not deplete

dopaminergic neurons in our substantia nigra cell culture, the

dramatic effect on neurite length suggested that these neurons were

undergoing a retrograde degeneration, as described for 6-OHDA-

induced cell death.

Subsequently, the substantia nigra cultures treated with 500 AM6-OHDAwere used to test the effect of a toxic stimulus on GDNF

levels. In these cultures, 100 AM H2O2 did not stimulate GDNF

expression, whereas in vehicle-treated cultures GDNF levels

increased to 156.01 T 0.65% of control (Fig. 7B). The basal levels

of GDNF in 6-OHDA treated cultures were reduced to 86.98 T3.60% of control cultures, suggesting that dopaminergic neurons

also play a tonic role in the expression of GDNF in our model.

Discussion

This study was undertaken to examine whether conditions

mimicking dopamine toxicity, proposed to occur in PD (Blum et al.,

2001; Lotharius and Brundin, 2002), altered GDNF expression in

the substantia nigra. The normal metabolism of dopamine produces

H2O2 that may be converted to highly reactive radicals, and the

oversupply of dopamine during therapeutic replacement with l-

DOPA might increase oxidative stress. We found that mild toxic

injury to dopaminergic neurons triggered GDNF up-regulation

involving soluble signals that induced GDNF expression in

astrocytes. We showed that the presence of still functional

dopaminergic neurons was important for injury-induced GDNF

up-regulation since this effect was abolished in cultures previously

treated with 6-OHDA.

In neuron–glia mixed cell cultures, l-DOPA (200 and 400

AM), but not H2O2 up to 150 AM, caused dopaminergic cell loss as

assessed by TH immunocytochemistry (Fig. 1B). However, the

effect of GDNF neutralization showed that H2O2 was indeed

damaging dopaminergic neurons since preventing GDNF from

properly interacting with its targets caused a reduction in

dopaminergic viability, whereas the GDNF antibody had no effect

on controls (Fig. 5). This observation suggests that the production

of GDNF protected dopaminergic neurons from the toxic effects of

H2O2. This agent was found to be toxic for mesencephalic neurons

cultured in the absence of astrocytes, but the lack of dopaminergic

cell loss in cultures treated with H2O2 was also observed by others

in a similar coculture model (Langeveld et al., 1995). The

protection by astrocytes might be due to glutathione (GSH) since

its concentrations in cultured neurons are relatively low, whereas

cultured astrocytes contain high levels of GSH (Raps et al., 1989;

Sagara et al., 1993) and can protect neurons against the toxicity of

H2O2 (Langeveld et al., 1995; Desagher et al., 1996). In this work

we show that astrocytes produce GDNF that can afford protection

to dopaminergic neurons against H2O2-induced toxicity.

Our data also suggest that the trophic effects of l-DOPA on

nigral dopaminergic neurons observed in some conditions (Mena

et al., 1997; Murer et al., 1998; Datla et al., 2001) might be

mediated, at least partly, by GDNF up-regulation. Previous studies

on a similar culture system reported that 50 AM l-DOPA had a

positive impact on dopaminergic cell viability (Mena et al., 1997),

but we did not observe any effect of 50 AM l-DOPA either on

dopaminergic cell viability (Fig. 1B) or on GDNF levels (Figs. 2A

and B). There are a number of possible explanations for this

difference: we used postnatal cell cultures from substantia nigra,

not from the total ventral midbrain, and midbrain astrocytes instead

of cortical ones. In addition, the incubation with l-DOPA was for

24 h, instead of 48 h.

Another finding of the present work was that GDNF levels

increased significantly in response to H2O2 (100 AM) or l-DOPA

(200 and 300 AM) in substantia nigra mixed cultures but not in

astrocyte cultures (Fig. 2), suggesting that neurons were playing a

role on GDNF up-regulation. They could be up-regulating GDNF

by themselves, or stimulating glial cells to increase GDNF

synthesis/release, or both. Since conditioned media from mixed

cultures treated with H2O2 or l-DOPA increased GDNF content in

astrocyte cultures (Fig. 6), and GDNF up-regulation in response

to H2O2 was abolished in cultures previously treated with 6-

OHDA (Fig. 7B), we propose that damaged, but still functional,

dopaminergic neurons release soluble mediators that act on

astrocytes leading to the up-regulation of GDNF as a neuro-

protective strategy. Although TH-positive cells were still present in

6-OHDA-treated substantia nigra cultures, they exhibited much

shorter processes than control cells, suggesting that dopaminergic

neurons were undergoing degeneration and were not functional any

more. These data highlight the delicate balance between an injured

cell, but still capable of signalling, and a no longer functional silent

cell.

The observation that 6-OHDA did not eliminate dopaminergic

neurons in the culture, at least under conditions that did not affect

the other cell types (Fig. 7A), is in agreement with results from

Bronstein et al. (1995) who found that the loss of TH-positive

neurons exposed to 6-OHDA was smaller in neuron–glia cultures

than in neuron-enriched cultures, although TH-positive processes

were severely pruned in both culture types. In contrast, a near

complete loss of dopaminergic neurons was reported in substantia

nigra postnatal cell cultures treated with 100 AM 6-OHDA,

whereas 500 AM 6-OHDA also caused a significant loss of

GABA-positive neurons (Ding et al., 2004). These discrepancies

are likely due to differences in the density of astrocytes since our

substantia nigra mixed cultures are prepared in two steps: first, a

confluent monolayer of midbrain astrocytes is established in

culture medium containing 10% calf serum, and then substantia

nigra neurons are plated on this feeding layer. Since the cultures

of Ding et al. (2004) were prepared in one step, and cells were

cultured in medium with only 2% serum, the density of astrocytes

was likely lower and therefore the protection afforded by

astrocytes against 6-OHDA toxicity was probably reduced. High

density of glial cells was also shown to confer protection to

dopaminergic neurons against MPP+ toxicity (Smeyne et al.,

2005).

GDNF expression was also shown to respond to injury in other

models, including nerve lesion (Trupp et al., 1995; Naveilhan et al.,

1997; Bar et al., 1998), ischemia (Miyazaki et al., 2001; Ikeda et al.,

2002) and mechanical injury to the spinal cord (Satake et al., 2000;

Widenfalk et al., 2001) or to the striatum (Liberatore et al., 1997).

Lesion of the nigrostriatal pathway in vivo by 6-OHDAwas found

to increase GDNF expression in the striatum (Zhou et al., 2000;

Nakajima et al., 2001; Yurek and Fletcher-Turner, 2001), but not in

the substantia nigra (Inoue et al., 1999; Yurek and Fletcher-Turner,

2001). In our model we found evidence indicating that dopami-

nergic neurons from the substantia nigra, in response to injury, may

trigger the expression of GDNF in local astrocytes as a neuro-

protective strategy. Substantial levels of GDNF mRNA were

observed in rat substantia nigra (Oo et al., 2005), thus supporting

the hypothesis of a local expression of GDNF. This trophic support

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542540

near neuron soma may be important when degeneration of

dopaminergic terminals in the striatum compromises the retrograde

transport of striatal GDNF.

An interesting observation of the present work was that only

intermediate concentrations of H2O2 or l-DOPA increased GDNF

levels, leading us to propose that mild but not strong injury to

dopaminergic neurons triggered GDNF up-regulation. We suggest

that the damage caused by the higher concentrations is probably

beyond the defensive ability of cells to set up a protective response.

Alternatively, the number of surviving dopaminergic neurons (only

60% upon treatment with 400 AM l-DOPA) may not be enough to

produce an effective concentration of mediator(s) of GDNF up-

regulation to act on astrocytes.

The observation that GDNF up-regulation in astrocyte cultures

incubated with conditioned media (20–30% above control) was

smaller than GDNF up-regulation in neuron–glia cultures (60–

70% above control) may be explained by several factors. The

concentration of the putative mediators may have decreased at the

24 h time point when the conditioned medium was collected. The

fast increase in GDNF mRNA in mixed cultures (Fig. 4) also

supports the hypothesis that the maximum concentration of the

hypothetical mediator(s) for GDNF up-regulation was reached well

before the 24 h time-point. This hypothesis was further supported

by the data showing that media conditioned for shorter periods

triggered larger increases in GDNF mRNA levels in astrocyte

cultures (Fig. 6B). In addition, the concentration of the mediator(s)

in conditioned media may not reflect the local concentrations

reached in mixed cultures where neurons and astrocytes are in

close contact.

The different temporal patterns observed for the increases in

GDNF mRNA levels upon treatment with H2O2 or l-DOPA (Fig.

4) may suggest that different mechanisms are involved. Since

H2O2 is known to directly activate transcription factors such as the

nuclear factor-kappa B (NF-nB) (Bowie and O’Neill, 2000), and

GDNF gene has a binding sequence for NF-nB (Baecker et al.,

1999), one may argue that the fast increase in GDNF mRNA could

be due to a direct effect of H2O2, rather than being mediated by

soluble factors released by damaged dopaminergic neurons.

However, if this was the case, we would expect to see up-

regulation of GDNF in astrocyte cultures incubated with H2O2.

The lack of direct effect of H2O2 on astrocytes may be due to the

fact that astrocytes have more glutathione peroxidase and catalase

activities, and a correspondingly greater capacity to metabolize

H2O2, than neurons (Makar et al., 1994; Desagher et al., 1996;

Dringen et al., 1999). In addition, the effect of GDNF neutraliza-

tion on dopaminergic cell viability in mixed cultures, and the effect

of conditioned media from H2O2-treated mixed cultures on GDNF

levels in astrocyte cultures suggest that damaged neurons signal

astrocytes to increase GDNF expression.

In both PD and experimental models of PD, the dramatic loss of

dopaminergic neurons is associated with a glial reaction composed

mainly of activated microglial cells and, to a lesser extent, of

reactive astrocytes. This glial response may be the source of

trophic factors and mediate protection against reactive oxygen

species (Teismann et al., 2003). The protective role of glial cells in

PD is supported by the fact that dopaminergic neurons degenerate

in areas with low density of astrocytes and are preserved in areas

with higher densities (Damier et al., 1993). This observation fits

our hypothesis that damaged dopaminergic neurons release

modulators that act on astrocytes to increase GDNF synthesis/

release. In areas with low density of glial cells, the probability of

the modulator to reach its target would be reduced and hence the

GDNF up-regulation would also be compromised.

In summary, our findings indicate that mild toxic damage to

dopaminergic neurons up-regulates GDNF expression in astro-

cytes, and that this response is mediated by soluble factors released

from dopaminergic neurons. Moreover, we show that up-regulation

of endogenous GDNF protects dopaminergic neurons from H2O2-

induced toxicity. To our knowledge, this is the first report on the

effect of l-DOPA on GDNF expression, and one of the very few

focusing on the effect of injury on GDNF levels in substantia nigra

(Inoue et al., 1999; Yurek and Fletcher-Turner, 2001). This has

special importance since GDNF has proven to be a potent factor for

protection of nigral dopaminergic neurons against toxin-induced

degeneration in vivo, and l-DOPA remains the drug of choice in

the symptomatic treatment of PD. Results from the recent

ELLDOPA clinical trial support the concept that l-DOPA does

not hasten the disease progression, but rather may slow down its

rate (Fahn, 2005). It is therefore tempting to relate this neuro-

protective effect with our observation that l-DOPA increased

GDNF levels. Understanding how GDNF expression and release

are regulated will be a valuable tool to the development of small

molecules targeted to regulatory check points that may prove

efficacious in the treatment of PD patients. The intercellular

mediators involved in the neuron–glia crosstalk upon dopaminer-

gic injury might constitute a therapeutic target aimed at up-

regulating neuroprotective factors.

Acknowledgments

We are grateful to Dr. David Sulzer (Columbia University, N.Y.,

USA) for his help in establishing the postnatal cell culture model

used in this work. This work was supported by Bissaya Barreto

Foundation and Foundation for Science and Technology, Portugal

(grant SFRH/BD/5337/2001 to Ana Saavedra).

References

Akerud, P., Canals, J.M., Snyder, E.Y., Arenas, E., 2001. Neuroprotection

through delivery of glial cell line-derived neurotrophic factor by neural

stem cells in a mouse model of Parkinson’s disease. J. Neurosci. 21,

8108–8118.

Baecker, P.A., Lee, W.H., Verity, A.N., Eglen, R.M., Johnson, R.M., 1999.

Characterization of a promoter for the human glial cell line-derived

neurotrophic factor gene. Brain. Res. Mol. Brain Res. 69, 209–222.

Bar, K.J., Saldanha, G.J., Kennedy, A.J., Facer, P., Birch, R., Carlstedt, T.,

Anand, P., 1998. GDNF and its receptor component Ret in injured

human nerves and dorsal root ganglia. NeuroReport 9, 43–47.

Blessing, H., Bareiss, M., Zettlmeisl, H., Schwarz, J., Storch, A., 2003.

Catechol-O-methyltransferase inhibition protects against 3,4-dihydrox-

yphenylalanine (DOPA) toxicity in primary mesencephalic cultures:

new insights into levodopa toxicity. Neurochem. Int. 42, 139–151.

Blin, J., Bonnet, A.M., Agid, Y., 1988. Does levodopa aggravate

Parkinson’s disease? Neurology 38, 1410–1416.

Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A.L., Sadoul, R.,

Verna, J.M., 2001. Molecular pathways involved in the neurotoxicity of

6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in

Parkinson’s disease. Prog. Neurobiol. 65, 135–172.

Bowenkamp, K.E., Hoffman, A.F., Gerhardt, G.A., Henry, M.A., Biddle,

P.T., Hoffer, B.J., Granholm, A.C., 1995. Glial cell line-derived

neurotrophic factor supports survival of injured midbrain dopaminergic

neurons. J. Comp. Neurol. 355, 479–489.

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542 541

Bowie, A., O’Neill, L.A., 2000. Oxidative stress and nuclear factor-kappaB

activation: a reassessment of the evidence in the light of recent

discoveries. Biochem. Pharmacol. 59, 13–23.

Bronstein, D.M., Perez-Otano, I., Sun, V., Mullis Sawin, S.B., Chan, J., Wu,

G.C., Hudson, P.M., Kong, L.Y., Hong, J.S., McMillian, M.K., 1995.

Glia-dependent neurotoxicity and neuroprotection in mesencephalic

cultures. Brain Res. 704, 112–116.

Burke, R.E., Antonelli, M., Sulzer, D., 1998. Glial cell line-derived

neurotrophic growth factor inhibits apoptotic death of postnatal

substantia nigra dopamine neurons in primary culture. J. Neurochem.

71, 517–525.

Chauhan, N.B., Siegel, G.J., Lee, J.M., 2001. Depletion of glial cell line-

derived neurotrophic factor in substantia nigra neurons of Parkinson’s

disease brain. J. Chem. Neuroanat. 21, 277–288.

Connor, B., Dragunow, M., 1998. The role of neuronal growth factors in

neurodegenerative disorders of the human brain. Brain Res. Brain Res.

Rev. 27, 1–39.

Damier, P., Hirsch, E.C., Zhang, P., Agid, Y., Javoy-Agid, F., 1993.

Glutathione peroxidase, glial cells and Parkinson’s disease. Neurosci-

ence 52, 1–6.

Datla, K.P., Blunt, S.B., Dexter, D.T., 2001. Chronic l-DOPA administra-

tion is not toxic to the remaining dopaminergic nigrostriatal neurons,

but instead may promote their functional recovery, in rats with partial 6-

OHDA or FeCl(3) nigrostriatal lesions. Mov. Disord. 16, 424–434.

Desagher, S., Glowinski, J., Premont, J., 1996. Astrocytes protect neurons

from hydrogen peroxide toxicity. J. Neurosci. 16, 2553–2562.

Diamond, S.G., Markham, C.H., 1990. Longitudinal study of effects of

early levodopa treatment on disability and mortality in Parkinson’s

disease. Adv. Neurol. 53, 399–403.

Ding, Y.M., Jaumotte, J.D., Signore, A.P., Zigmond, M.J., 2004. Effects of

6-hydroxydopamine on primary cultures of substantia nigra: specific

damage to dopamine neurons and the impact of glial cell line-derived

neurotrophic factor. J. Neurochem. 89, 776–787.

Dringen, R., Kussmaul, L., Gutterer, J.M., Hirrlinger, J., Hamprecht, B.,

1999. The glutathione system of peroxide detoxification is less efficient

in neurons than in astroglial cells. J. Neurochem. 72, 2523–2530.

Du, F., Li, R., Huang, Y., Li, X., Le, W., 2005. Dopamine D3 receptor-

preferring agonists induce neurotrophic effects on mesencephalic

dopamine neurons. Eur. J. Neurosci. 22, 2422–2430.

Fahn, S., 2005. Does levodopa slow or hasten the rate of progression of

Parkinson’s disease? J. Neurol. 252 (Suppl. 4), IV37–IV42.

Gash, D.M., Zhang, Z., Ovadia, A., Cass, W.A., Yi, A., Simmerman, L.,

Russell, D., Martin, D., Lapchak, P.A., Collins, F., Hoffer, B.J.,

Gerhardt, G.A., 1996. Functional recovery in parkinsonian monkeys

treated with GDNF. Nature 380, 252–255.

Gill, S.S., Patel, N.K., Hotton, G.R., O’Sullivan, K., McCarter, R.,

Bunnage, M., Brooks, D.J., Svendsen, C.N., Heywood, P., 2003. Direct

brain infusion of glial cell line-derived in Parkinson disease. Nat. Med.

9, 589–595.

Gille, G., Rausch, W.D., Hung, S.T., Moldzio, R., Ngyuen, A., Janetzky,

B., Engfer, A., Reichmann, H., 2002. Protection of dopaminergic

neurons in primary culture by lisuride. J. Neural Transm. 109,

157–169.

Grondin, R., Zhang, Z., Yi, A., Cass, W.A., Maswood, N., Andersen, A.H.,

Elsberry, D.D., Klein, M.C., Gerhardt, G.A., Gash, D.M., 2002.

Chronic, controlled GDNF infusion promotes structural and functional

recovery in advanced parkinsonian monkeys. Brain 125, 2191–2201.

Gwinn-Hardy, K., Evidente, V.G., Waters, C., Muenter, M.D., Hardy, J.,

1999. l-dopa slows the progression of familial parkinsonism. Lancet

353, 1850–1851.

Hoffer, B.J., Hoffman, A., Bowenkamp, K., Huettl, P., Hudson, J., Martin,

D., Lin, L.F., Gerhardt, G.A., 1994. Glial cell line-derived neurotrophic

factor reverses toxin-induced injury to midbrain dopaminergic neurons

in vivo. Neurosci. Lett. 182, 107–111.

Hurelbrink, C.B., Barker, R.A., 2001. Prospects for the treatment of

Parkinson’s disease using neurotrophic factors. Expert Opin. Pharmac-

other. 2, 1531–1543.

Ikeda, T., Koo, H., Xia, Y.X., Ikenoue, T., Choi, B.H., 2002. Bimodal

upregulation of glial cell line-derived neurotrophic factor (GDNF) in the

neonatal rat brain following ischemic/hypoxic injury. Int. J. Dev.

Neurosci. 20, 555–562.

Inoue, T., Tsui, J., Wong, N., Wong, S.Y., Suzuki, F., Kwok, Y.N., 1999.

Expression of glial cell line-derived neurotrophic factor and its mRNA

in the nigrostriatal pathway following MPTP treatment. Brain Res. 826,

306–308.

Kearns, C.M., Gash, D.M., 1995. GDNF protects nigral dopamine neurons

against 6-hydroxydopamine in vivo. Brain Res. 672, 104–111.

Kordower, J.H., Emborg, M.E., Bloch, J., Ma, S.Y., Chu, Y., Leventhal, L.,

McBride, J., Chen, E.Y., Palfi, S., Roitberg, B.Z., Brown, W.D.,

Holden, J.E., Pyzalski, R., Taylor, M.D., Carvey, P., Ling, Z., Trono, D.,

Hantraye, P., Deglon, N., Aebischer, P., 2000. Neurodegeneration

prevented by lentiviral vector delivery of GDNF in primate models of

Parkinson’s disease. Science 290, 767–773.

Kozlowski, D.A., Connor, B., Tillerson, J.L., Schallert, T., Bohn, M.C.,

2000. Delivery of a GDNF gene into the substantia nigra after a

progressive 6-OHDA lesion maintains functional nigrostriatal connec-

tions. Exp. Neurol. 166, 1–15.

Langeveld, C.H., Jongenelen, C.A., Schepens, E., Stoof, J.C., Bast, A.,

Drukarch, B., 1995. Cultured rat striatal and cortical astrocytes protect

mesencephalic dopaminergic neurons against hydrogen peroxide toxic-

ity independent of their effect on neuronal development. Neurosci. Lett.

192, 13–16.

Liberatore, G.T., Wong, J.Y., Porritt, M.J., Donnan, G.A., Howells, D.W.,

1997. Expression of glial cell line-derived neurotrophic factor (GDNF)

mRNA following mechanical injury to mouse striatum. NeuroReport 8,

3097–3101.

Lin, L.F., Doherty, D.H., Lile, J.D., Bektesh, S., Collins, F., 1993. GDNF: a

glial cell line-derived neurotrophic factor for midbrain dopaminergic

neurons. Science 260, 1130–1132.

Lotharius, J., Brundin, P., 2002. Pathogenesis of Parkinson’s disease:

dopamine, vesicles and alpha-synuclein. Nat. Rev., Neurosci. 3,

932–942.

Lyras, L., Zeng, B.Y., McKenzie, G., Pearce, R.K., Halliwell, B., Jenner, P.,

2002. Chronic high dose l-DOPA alone or in combination with the

COMT inhibitor entacapone does not increase oxidative damage or

impair the function of the nigro-striatal pathway in normal cynomologus

monkeys. J. Neural Transm. 109, 53–67.

Makar, T.K., Nedergaard, M., Preuss, A., Gelbard, A.S., Perumal, A.S.,

Cooper, A.J., 1994. Vitamin E, ascorbate, glutathione, glutathione

disulfide, and enzymes of glutathione metabolism in cultures of chick

astrocytes and neurons: evidence that astrocytes play an important role

in antioxidative processes in the brain. J. Neurochem. 62, 45–53.

Markham, C.H., Diamond, S.G., 1986. Long-term follow-up of early dopa

treatment in Parkinson’s disease. Ann. Neurol. 19, 365–372.

Melamed, E., Rosenthal, J., Globus, M., Cohen, O., Uzzan, A., 1985.

Suppression of MPTP-induced dopaminergic neurotoxicity in mice by

nomifensine and l-DOPA. Brain Res. 342, 401–404.

Mena, M.A., Pardo, B., Casarejos, M.J., Fahn, S., Garcia, D.Y., 1992.

Neurotoxicity of levodopa on catecholamine-rich neurons. Mov. Disord.

7, 23–31.

Mena, M.A., Davila, V., Sulzer, D., 1997. Neurotrophic effects of l-DOPA

in postnatal midbrain dopamine neuron/cortical astrocyte cocultures.

J. Neurochem. 69, 1398–1408.

Miyazaki, H., Nagashima, K., Okuma, Y., Nomura, Y., 2001. Expression of

glial cell line-derived neurotrophic factor induced by transient forebrain

ischemia in rats. Brain Res. 922, 165–172.

Mogi, M., Togari, A., Kondo, T., Mizuno, Y., Kogure, O., Kuno, S.,

Ichinose, H., Nagatsu, T., 2001. Glial cell line-derived neurotrophic

factor in the substantia nigra from control and parkinsonian brains.

Neurosci. Lett. 300, 179–181.

Murer, M.G., Dziewczapolski, G., Menalled, L.B., Garcia, M.C., Agid, Y.,

Gershanik, O., Raisman-Vozari, R., 1998. Chronic levodopa is not toxic

for remaining dopamine neurons, but instead promotes their recovery, in

rats with moderate nigrostriatal lesions. Ann. Neurol. 43, 561–575.

A. Saavedra et al. / Neurobiology of Disease 23 (2006) 533–542542

Mytilineou, C., Walker, R.H., JnoBaptiste, R., Olanow, C.W., 2003.

Levodopa is toxic to dopamine neurons in an in vitro but not an in

vivo model of oxidative stress. J. Pharmacol. Exp. Ther. 304, 792–800.

Nakajima, K., Hida, H., Shimano, Y., Fujimoto, I., Hashitani, T., Kumazaki,

M., Sakurai, T., Nishino, H., 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.

Naveilhan, P., ElShamy, W.M., Ernfors, P., 1997. Differential regulation of

mRNAs for GDNF and its receptors Ret and GDNFR alpha after sciatic

nerve lesion in the mouse. Eur. J. Neurosci. 9, 1450–1460.

Oo, T.F., Ries, V., Cho, J., Kholodilov, N., Burke, R.E., 2005. Anatomical

basis of glial cell line-derived neurotrophic factor expression in the

striatum and related basal ganglia during postnatal development of the

rat. J. Comp. Neurol. 484, 57–67.

Perry, T.L., Yong, V.W., Ito, M., Foulks, J.G., Wall, R.A., Godin, D.V.,

Clavier, R.M., 1984. Nigrostriatal dopaminergic neurons remain

undamaged in rats given high doses of l-DOPA and carbidopa

chronically. J. Neurochem. 43, 990–993.

Raps, S.P., Lai, J.C., Hertz, L., Cooper, A.J., 1989. Glutathione is present in

high concentrations in cultured astrocytes but not in cultured neurons.

Brain Res. 493, 398–401.

Rosenblad, C., Martinez-Serrano, A., Bjorklund, A., 1998. Intrastriatal glial

cell line-derived neurotrophic factor promotes sprouting of spared

nigrostriatal dopaminergic afferents and induces recovery of function in

a rat model of Parkinson’s disease. Neuroscience 82, 129–137.

Saavedra, A., Baltazar, G., Carvalho, C.M., Duarte, EP., 2005. GDNF

modulates HO-1 expression in substantia nigra postnatal cell cultures.

Free Radical Biol. Med. 39, 1611–1619.

Sagara, J.I., Miura, K., Bannai, S., 1993. Maintenance of neuronal

glutathione by glial cells. J. Neurochem. 61, 1672–1676.

Sarabi, A., Hoffer, B.J., Olson, L., Morales, M., 2001. GFRalpha-1 mRNA

in dopaminergic and nondopaminergic neurons in the substantia nigra

and ventral tegmental area. J. Comp. Neurol. 441, 106–117.

Satake, K., Matsuyama, Y., Kamiya, M., Kawakami, H., Iwata, H., Adachi,

K., Kiuchi, K., 2000. Up-regulation of glial cell line-derived neuro-

trophic factor (GDNF) following traumatic spinal cord injury. Neuro-

Report 11, 3877–3881.

Sauer, H., Rosenblad, C., Bjorklund, A., 1995. Glial cell line-derived

neurotrophic factor but not transforming growth factor beta 3 prevents

delayed degeneration of nigral dopaminergic neurons following

striatal 6-hydroxydopamine lesion. Proc. Natl. Acad. Sci. U. S. A.

92, 8935–8939.

Siegel, G.J., Chauhan, N.B., 2000. Neurotrophic factors in Alzheimer’s and

Parkinson’s disease brain. Brain Res. Brain Res. Rev. 33, 199–227.

Smeyne, M., Smeyne, R.J., 2002. Method for culturing postnatal substantia

nigra as an in vitro model of experimental Parkinson’s disease. Brain

Res. Brain Res. Protoc. 9, 105–111.

Smeyne, M., Jiao, Y., Shepherd, K.R., Smeyne, R.J., 2005. Glia cell

number modulates sensitivity to MPTP in mice. Glia 52, 144–152.

Soto-Otero, R., Mendez-Alvarez, E., Hermida-Ameijeiras, A., Munoz-

Patino, A.M., Labandeira-Garcia, J.L., 2000. Autoxidation and neuro-

toxicity of 6-hydroxydopamine in the presence of some antioxidants:

potential implication in relation to the pathogenesis of Parkinson’s

disease. J. Neurochem. 74, 1605–1612.

Teismann, P., Tieu, K., Cohen, O., Choi, D.K., Wu, D.C., Marks, D., Vila,

M., Jackson-Lewis, V., Przedborski, S., 2003. Pathogenic role of glial

cells in Parkinson’s disease. Mov. Disord. 18, 121–129.

Tomac, A., Lindqvist, E., Lin, L.F., Ogren, S.O., Young, D., Hoffer, B.J.,

Olson, L., 1995. Protection and repair of the nigrostriatal dopaminergic

system by GDNF in vivo. Nature 373, 335–339.

Trupp, M., Ryden, M., Jornvall, H., Funakoshi, H., Timmusk, T., Arenas,

E., Ibanez, C.F., 1995. Peripheral expression and biological activities of

GDNF, a new neurotrophic factor for avian and mammalian peripheral

neurons. J. Cell Biol. 130, 137–148.

Trupp, M., Belluardo, N., Funakoshi, H., Ibanez, C.F., 1997. Complemen-

tary and overlapping expression of glial cell line-derived neurotrophic

factor (GDNF), c-ret proto-oncogene, and GDNF receptor-alpha

indicates multiple mechanisms of trophic actions in the adult rat CNS.

J. Neurosci. 17, 3554–3567.

Uitti, R.J., Ahlskog, J.E., Maraganore, D.M., Muenter, M.D., Atkinson,

E.J., Cha, R.H., O’Brien, P.C., 1993. Levodopa therapy and survival in

idiopathic Parkinson’s disease: Olmsted County project. Neurology 43,

1918–1926.

Wagner, J., Akerud, P., Castro, D.S., Holm, P.C., Canals, J.M., Snyder, E.Y.,

Perlmann, T., Arenas, E., 1999. Induction of a midbrain dopaminergic

phenotype in Nurr1-overexpressing neural stem cells by type 1

astrocytes. Nat. Biotechnol. 17, 653–659.

Widenfalk, J., Lundstromer, K., Jubran, M., Brene, S., Olson, L.,

2001. Neurotrophic factors and receptors in the immature and adult

spinal cord after mechanical injury or kainic acid. J. Neurosci. 21,

3457–3475.

Winkler, C., Sauer, H., Lee, C.S., Bjorklund, A., 1996. Short-term GDNF

treatment provides long-term rescue of lesioned nigral dopaminergic

neurons in a rat model of Parkinson’s disease. J. Neurosci. 16,

7206–7215.

Yurek, D.M., Fletcher-Turner, A., 2001. Differential expression of GDNF,

BDNF, and NT-3 in the aging nigrostriatal system following a

neurotoxic lesion. Brain Res. 891, 228–235.

Zhou, J., Yu, Y., Tang, Z., Shen, Y., Xu, L., 2000. Differential expression of

mRNAs of GDNF family in the striatum following 6-OHDA-induced

lesion. NeuroReport 11, 3289–3293.

Zigmond, M.J., Hastings, T.G., Perez, R.G., 2002. Increased dopamine

turnover after partial loss of dopaminergic neurons: compensation or

toxicity? Parkinsonism Relat. Disord. 8, 389–393.