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Introduction
Glial cell line-derived neurotrophic factor (GDNF) was
first isolated by virtue of its ability to induce dopamine (DA)
uptake and cell survival in culture of embryonic ventral
midbrain DA cells. Further experimental results have also
revealed that GDNF may protect DA cells from injury by
toxicity[1,2]. Although the biological effects of GDNF on DA cells
have been studied extensively, the mechanisms underlying
the role of GDNF are less known.
GDNF signals via multicomponent receptors that consist
of the Ret receptor tyrosine kinase plus a
glycosylphosphati-dylinositol-linked coreceptor named GDNF family receptor
a1 (GFRa1). The binding of GDNF to Ret and GFRa1 induces
Ret phosphorylation[3,4]. After phosphorylation, Ret induces
the activation of several intracellular pathways, among which
the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) pathway
is of particular interest[5].
The PI3-K/Akt pathway is an important regulator of
neuronal survival, both in central and peripheral nervous
systems[6]. The PI3-K/Akt pathway is initiated by the activation
of PI3-K, which in turn activates a cascade of downstream
effectors including the serine/threonine kinase
Akt[7]. The survival of sympathetic neurons of the superior cervical
ganglion (SCG) induced by nerve growth factor (NGF) is
critically dependent upon an intact PI3-K/Akt
pathway[8]. GDNF is also able to activate the PI3-K/Akt pathway and promote
the survival of SCG[9]. However, whether the PI3K/Akt
pathway is involved in the survival/differentiation effects of
GDNF on primary cultured DA cells is not yet well understood.
Further studies show that both GFRa1 and Ret are present in
midbrain DA cells[10].
In the present study, we examine the intracellular
pathways activated by GDNF in DA cells in
vitro, and whether the PI3-K/Akt pathway contributes to GDNF-induced DA
cells survival/differentiation.
Materials and methods
Cell culture Primary DA cell culture was established
from the ventral mesencephalic tissues of rat embryos as
described previously[10]. Briefly, Sprague-Dawley pregnant
rats were deeply anesthetized on gestational d 18, and fetuses were rapidly removed from the uterus and transferred
to ice-cold Dulbecco's modified Eagle's medium (DMEM).
The mesencephalic flexure enriched with DA cells was cut
off from the fetal brain and minced into 1 mm×1 mm×1 mm
pieces. After incubation for 15 min at 37
oC with 0.25% trypsin and 0.02% EDTA solution, the cells were separated by
trituration through a syringe and passed through a 150 mesh
sieve. The cell suspension was centrifuged for 5 min and
then resuspended in complete medium [DMEM/F12 1:1,
containing 10% fetal bovine serum (FBS), 4 mmol/L glutamine,
100 U/mL penicillin G sodium, and 100 µg/mL
streptomycin sulfate]. The cells were plated at a density of
1.5×105 cells/well onto 24 well plates, which were pre-coated with 0.1 g/L poly-L-lysine for morphological or Western blot
analysis, respectively. After 24 h in culture (1DIV), the
media were replaced with serum-free medium
(NeurobasalTM medium containing 2% B27 supplement, 4 mmol/L glutamine,
100 U/mL penicillin G sodium, 100 µg/mL streptomycin
sulfate) and 10 ng/mL GDNF with or without wortmannin
(Calbiochem, KY 12420, Germany). Wortmannin was used at
50 nmol/L, then on every other day, half of the same medium
was replaced. On 6DIV, TH immunostaining was processed.
Cultures were maintained at 37 oC in an atmosphere of 5%
CO2/95% air and 100% relative humidity.
Tissue culture We used the embryonic d 18
Sprague-Dawley rats in this study. The pregnant Sprague-Dawley
rats were deeply anesthetized, and the fetuses were rapidly
removed from the uterus and transferred to ice-cold DMEM.
The whole brain was rapidly removed and immediately chilled
for 3_5 min in ice-cold DMEM, which had been
preoxygen-ated in a 95% O2/5%
CO2 incubator. The brains were then embedded in low-melting point agarose [2.5% in
phosphate-buffered saline (PBS); type VII agarose, A9045; Sigma, St
Louis, MO, USA], mounted onto the McIlwain tissue
chopper stage. Coronal sections (400 µm) were cut, followed by
separation in ice-cold DMEM, supplemented with 10% FBS
and penicillin-streptomycin (100 U/mL, 100 µg/mL,
respectively). Slices of interest were transferred onto 30 mm
Millicell-CM (Millipore, Bedford, MA, USA) culture plate
inserts (0.4 µm, 4 per well) in 6-well tissue culture plates
containing 1.5 mL of the above growth medium. Slices
containing a clearly defined midbrain(identified using the atlas
of Paxinos and Watson 1986[11]) were used for the experiments.
The control and treatment groups for a single trial were
prepared at the same time and cultured for the same durations.
Throughout their growth period, the slices were kept at
an interface between the growth medium and the humid
atmosphere. The cultures were maintained at 37 °C in a
humidified atmosphere of 5% CO2/95%
O2. After 24 h in the culture (1DIV), the culture media were replaced with a
serum-free medium (NeurobasalTM medium containing 2% B27
supplement, 4 mmol/L glutamine, 100 U/mL penicillin G
sodium, and 100 µg/mL streptomycin sulfate). Two hundred
ng/mL GDNF with or without wortmannin was added. Two
hundred nmol/L wortmannin was added 1 h prior to the GDNF
addition. Then on every other day, half of the same medium
was replaced. On 6DIV, immunohistochemistry and Western
blotting were performed to detect tyrosine hydroxylase (TH).
To study the activation of the PI3-K/Akt pathways, the
slices were cultured in serum-free medium. On 6DIV,
Two hundred ng/mL GDNF with or without wortmannin was
added. Akt phosphorylation was examined 30 min after GDNF
was added.
Immunostaining On 6DIV, thin paraffin sections were
deparaffinized and rehydrated, and the cells were fixed in 4%
paraformaldehyde for 20 min. To block residual endogenous
peroxidase activity, the sections and cells were incubated
for 10 min with 3% hydrogen peroxide in PBS. After being
washed 3 times with PBS for 5 min each, they were blocked
with 1% bovine serum albumin (BSA) in PBS for 1 h at 37 °C,
washed with PBS, and incubated with monoclonal mouse
anti-rat tyrosine hydroxylase (TH) antibody at 1:3000 (Sigma,
USA) overnight at 4 °C. After being washed 3 times with
PBS, they were incubated with a biotinylated goat anti-mouse
IgG (1:50; Sigma, USA) overnight at 4 °C.
Peroxidase-conjugated streptavidin was added for 30 min at room temperature
(RT) and the cultures were stained for peroxidase reaction
by incubation with a mixture of diaminobenzidine and
hydrogen peroxide for 5_10 min. The slides and cells were
cleared and mounted with a microscope. Controls were
prepared without the primary antibody.
To study the activation of the PI3-K/Akt pathway in our
culture models, TH/p-Akt immunofluorescence double stain
was processed. On 6DIV, the cells were treated as described
earlier, and incubated with monoclonal mouse anti-rat TH
antibody 1:3000 and monoclonal rabbit anti-mouse p-Akt
antibody 1:1000 overnight at 4 °C. After being washed 3
times with PBS, they were incubated with Cy3-conjugated
goat anti-mouse IgG or fluorescein(FITC) -conjugated goat
anti-rabbit IgG for 2 h at 37 °C. After being washed 3 times
with PBS, they were observed with a confocal microscope.
Western blotting After GDNF exposure on 6DIV, slice
tissues were collected rapidly in ice-cold PBS, then
homogenized at 4 °C in ice-cold lysis buffer [10 mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.9, 0.5
mmol/L MgCl2, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1
mmol/L O,O'-Bis(2-aminoethyl)ethyleneglycol-
N,N,N',N'-tetraacetic acid, 50 mmol/L NaF, 5 mmol/L dithiothreitol, 10 mmol/L
phosphoglycerol, 1 mmol/L
Na3VO4, 1% NP40, 1 mmol/L benzamidine and enzyme inhibitors: 5 mg/mL
phenylmethyl-sulfonyl fluoride, and 5 mg/mL each of pepstatin A, leupeptin,
aprotinin]. After centrifugation, the supernatants were stored
at -80 °C. Equal amounts (50 µg) of protein were separated
by 10% SDS-PAGE, electrotransferred onto a nitrocellulose
membrane and immunoblotted. Mouse anti-rat TH antibody
(Sigma, USA) was used at 1:1000 dilution. Rabbit anti-mouse
p-Akt antibody (Cell Signaling Technology, Beverly, MA,
USA) was used at 1:2000 dilution. Goat anti-mouse AP
(Sigma, USA) or goat anti-rabbit AP (Sigma, USA) as a
secondary antibody were used at 1:5000 dilution. The negative
control was prepared without the primary antibody, but
including all other procedures. After blotting, the bands on
the filter were scanned and analyzed with an image analyzer
(LabWorks Software, UVP Upland, CA, USA). The optical
density of the band in each lane was expressed as `fold'
versus that in the sham control lane in the same filter. To
standardize the total protein content in each lane, membranes
were incubated at RT with a mouse monoclonal antibody
against pan-Akt (1:2000; Cell Signaling Technology, USA)
for 1 h. Other procedures were the same as described earlier.
Data analysis The effects of GDNF on DA neuronal
survival/differentiation, and the impact of the inhibition of
PI3-K/Akt pathway on the actions of GDNF, were measured and
quantified. First, the number of TH-ir cells per
mm2 and the number of primary neurite of 60 randomly selected DA cells
were used as the index of DA cell survival/differentiation.
The DA cells were selected randomly in the right-up, left-up,
right-down, left-down and central part of the different visual
field, respectively. Second, the phosphorylation of
p-Akt was used as an index of the activation of the
PI3-K/Akt path-way. Results were compared by one-way ANOVA test using
the SigmaStat32 statistical program.
Results
GDNF promoted the survival/differentiation of midbrain
DA cells In the present study, both midbrain slice culture
and cell culture, in which GDNF could promote the
survival/differentiation of DA cells, were established as our
experimental models. In each model, the identity of the DA cells in
the culture was confirmed by the positive staining for TH.
The first step of our study was to decide the appropriate
concentration of GDNF for the effective biological effect to
promote the survival/differentiation of DA cells. In the cell
culture model, the concentration of GDNF was 10 ng/mL,
which is consistent with Horger's
report[12], while in the slice culture model the concentration was 200 ng/mL, which is
consistent with our previous study.
Consistent with previous
findings[1,13,14], the present results showed that GDNF promoted the survival and
morphological differentiation of DA cells in the 2 culture models.
The results of immunostaining showed that the number of
TH-ir cells per mm2 in the GDNF-treated group
(18.63±0.95) was significantly more than that in the control of the slice
culture (8.76±0.75; Figure 1), and that both the number of
TH-ir cells per mm2 and the neurite number of TH-ir cells in
the GDNF-treated group (6.01±0.43 and 4.89±0.46) were
significantly more than that of the control of the cell culture
(3.65±0.88 and 2.49±0.42; n=6; Figure 2).
The results of Western blotting also showed that the
level of TH expression in the GDNF-treated group was
significantly higher than that of the control
(n=3; Figure 3).
The PI3-K/Akt pathway was activated when GDNF
exerted the survival/differentiation effect on DA
cells We then explored the possible intracellular pathways
underlying the effect of GDNF on DA cells. It had been
demonstrated that the binding of GDNF to its receptors initiated
several intracellular pathways, among which the PI3-K/Akt
pathway was of particular interest. To test whether GDNF
was able to activate the PI3-K/Akt pathway in our
experimental models, the cells in the 2 cultures were stimulated
with GDNF (10 ng/mL or 200 ng/mL) at 6DIV. Thirty minutes
after the addition of GDNF into the media, the
phosphorylation of Akt was examined by TH/p-Akt immunofluorescence
double staining and by Western blotting. The results showed
that p-Akt was expressed in the TH-ir cells (Figure 4) and the
level of p-Akt expression in the GDNF-treated group was
higher than that of the control (Figure 5). It was suggested
that the PI3-K/Akt pathway was activated after GDNF
treat-ment.
Survival/differentiation effect of GDNF on DA cells was
abolished by the inhibitor of the PI3-K/Akt pathway
Wortmannin was used to specifically block the PI3-K/Akt
pathway. To further demonstrate the role of the PI3-K/Akt
pathway in the survival/differentiation effect of GDNF on
DA cells, GDNF (10 ng/mL or 200 ng/mL) with or without
wortmannin was added to cultures at 1DIV. Wortmannin
was plused into the medium 1 h before GDNF. Then on
6DIV, TH immunostaining and Western blotting were performed. The result of TH immunostaining showed that
wortmannin not only blocked the phosphorylation of Akt
induced by GDNF, but also abolished the effect of GDNF on
neuronal survival/differentiation of DA cells. The number of
TH-ir cells per mm2 in the PI3-K/Akt pathway-inhibited group
(6.98±0.58) was significantly lower than that of the
GDNF-treated group (18.63±0.95) in the slice culture (Figure 1). The
number of TH_ir cells per mm2 and the neurite number of
TH_ir cells in the PI3-K/Akt pathway-inhibited group (3.79±0.62 and 2.50±0.25) was significantly lower than that of the
GDNF-treated group (6.01±0.43 and 4.89±0.46) in the cell
culture (n=6; Figure 2). Western blot analysis of TH
expression in such conditions confirmed the above results
(n=3; Figure 3). The expression of TH decreased in the PI3-K/Akt
pathway-inhibited group. These results suggest that the
PI3-K/Akt pathway mediates the survival/differentiation
effects of GDNF on DA cells.
Discussion
The study was conducted both on the cell culture and
on the slice culture of the midbrain. The role of the
PI3-K/Akt pathway in mediating the effect of GDNF on DA cells
was explored. Our results showed that the PI3-K/Akt
pathway was activated when GDNF promoted the
survival/differentiation of DA cells; when the PI3-K/Akt pathway was
blocked by wortmannin, the effect of GDNF on DA cells was
abolished. The results suggest that the PI3-K/Akt pathway
may be involved in mediating the survival/differentiation role
of GDNF on DA cells.
Through binding to its receptors, GDNF may induce the
activation of the extracellular regulated kinase
(ERK)-mitogen-activated protein kinase (MAPK) and the PI3-K/Akt
pathway[5]. The PI3-K/Akt pathway has been implicated in
the survival-promoting
mechanisms[15_18].
In the present work, we show that GDNF increases the
phosphorylation of Akt, indicating that the PI3-K/Akt
pathway is activated. To confirm the involvement of the
PI3-K/Akt pathway in mediating the effect of GDNF, we treated
cultured cells with wortmannin, which is the inhibitor of the
PI3-K/Akt pathway. The results showed that when
wortman-nin was added to the medium, the number of
TH-ir cells and the neurite number of TH-ir cells dramatically decreased
compared with the control, suggesting that the PI3-K/Akt
pathway might play a striking role in mediating the
survival/differentiation effect of GDNF on cultured DA cells. This is
consistent with previous observations that the PI3-K/Akt
pathway was found to mediate the survival effect of GDNF
on cultured serum-starved spinal motor neurons, sympathetic
neurons, and cerebellar granule
cells[19]. Moreover, the role of the PI3-K/Akt pathway as a mediator of the trophic effect
of several trophic factors has been described previously in
the brain-derived neurotrophic factor-mediated survival of
cultured cerebellar granule neurons[20] or spinal cord
medial terminal nuclei[18], in NGF maintained PC12 or SGC
cells[8,16], and in cerebellar granule neurons maintained with
Insulin-like growth factor I[15,17]. However, the present work
demonstrated the involvement of the PI3-K/Akt pathway in
mediating the survival/differentiation process of GDNF on DA cells
both in cell culture and in slice culture.
Opinions about the ERK-MAPK pathway have been perplexing. Some studies consider that both the PI3-K/Akt
pathway and the ERK-MAPK pathway play a role in cell
survival[21,22], while others think that the activation of the
ERK_MAPK pathway is not involved in the cellular events
directly related with cell survival. However, the activation of
this pathway will be an important step in mediating neuronal
differentiation[23]. It seems that different growth factors
acting on different cell type may have different mechanisms to
achieve certain actions; that further studies need to be
conducted to uncover the real sense of these mechanisms.
Survival signals from various cell surface receptors
activate PI3-K to phosphorylate the downstream effector Akt,
which plays key roles in cellular processes such as glucose
metabolism, cell proliferation, apoptosis, transcription, and
cell migration. The exact role of activated Akt is determined
by its downstream target. So the elucidation of the
anti-apoptotic function of Akt signaling immediately precipitated
an intensive search for downstream targets involved in cell
survival. One possible downstream target on which this
signaling cascade converges is transcription factor nuclear
factor-kB (NF-kB). Upon the activation of Akt,
NF-kB may activate the transcription of anti-apoptotic proteins such as
the inhibitor of apoptosis proteins, c-IAP1 and c-IAP2.
In conclusion, our work demonstrated that the
activation of the PI3-K pathway is involved in the effect of GDNF
on cultured DA cells in both cell culture and slice culture
models. The PI3-K/Akt pathway plays a pivotal role in DA
neuronal survival/differentiation after GDNF stimulation.
Gaining insight into the cellular mechanisms underlying the
effects of GDNF may reveal cellular targets for treating
Parkinson's disease.
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