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Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor
Accepted Manuscript Title: Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor Authors: Hidekazu Sotoyama, Yuriko Iwakura, Kanako Oda, Toshikuni Sasaoka, Nobuyuki Takei, Akiyoshi Kakita, Hideki Enomoto, Hiroyuki Nawa PII: DOI: Reference:
S0304-3940(17)30485-8 http://dx.doi.org/doi:10.1016/j.neulet.2017.06.005 NSL 32886
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
4-4-2017 8-6-2017 8-6-2017
Please cite this article as: Hidekazu Sotoyama, Yuriko Iwakura, Kanako Oda, Toshikuni Sasaoka, Nobuyuki Takei, Akiyoshi Kakita, Hideki Enomoto, Hiroyuki Nawa, Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sotoyama et al. NSL-17600.R2
Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor
Hidekazu Sotoyama1, Yuriko Iwakura1, Kanako Oda2, Toshikuni Sasaoka2 Nobuyuki Takei1, Akiyoshi Kakita3, Hideki Enomoto4, Hiroyuki Nawa1
1. Department of Molecular Neurobiology, Brain research institute, Niigata University 2. Department of Comparative and Experimental Medicine, Brain Research Institute,, Niigata University 3. Department of Pathology, Brain Research Institute, Niigata University 4. Laboratory for Neural Differentiation and Regeneration, Graduate School of Medicine, Kobe University
Correspondence to: Hiroyuki Nawa Department of Molecular Biology, Brain Research Institute Niigata University, Asahimachi-dori 1-757, Niigata 951-8585, Japan Tel: +81-25-227-0613, Fax: +81-25-227-0815, E-mail: [email protected]
1
HIGHLIGHTS >The expression of GDNF transgene increases RET phosphorylation in the brain. >The chronic increase in glial GDNF reduces the dopamine content and tyrosine hydroxylase activity. > Extracellular concentrations of dopamine and its metabolism decrease in the striatum of transgenic mice. > The alteration of locomotor traits of transgenic mice is consistent with dopaminergic changes.
Abstract
Glial cell line-derived neurotrophic factor (GDNF) positively regulates the development and
maintenance of in vitro dopaminergic neurons. However, the in vivo influences of GDNF signals on
the brain dopamine system are controversial and not fully defined. To address this question, we
analyzed dopaminergic phenotypes of the transgenic mice that overexpress GDNF under the control
of the glial Gfap promoter in the brain. Compared with wild-type, the GDNF transgenic mice
contained higher levels of GDNF protein and phosphorylated RET receptors in the brain. However,
there were reductions in the levels of tyrosine hydroxylase (TH), dopamine, and its metabolite
homovanillic acid in the striatum of transgenic mice. The TH reduction appeared to occur during
postnatal development. Immunohistochemistry revealed that striatal TH density was reduced in
transgenic mice with no apparent signs of neurodegeneration. In agreement with these
neurochemical traits, basal levels of extracellular dopamine and high K+-induced dopamine efflux
were decreased in the striatum of transgenic mice. We also explored the influences of GDNF
overexpression on lomomotor behavior. GDNF transgenic mice exhibited lower stereotypy and
rearing in a novel environment compared with wild-type mice. These results suggest that chronic 2
overexpression of GDNF in brain astrocytes exerts an opposing influence on nigrostriatal dopamine
metabolism and neurotransmission.
Keywords: dopamine, GDNF, RET, tyrosine hydroxylase, neurotrophic factor 1. Introduction
Glial cell line-derived neurotrophic factor (GDNF) belongs to the transforming growth
factor-beta (TGF) superfamily whose members include neurturin, artemin, persephin, and others [1]. These neurotrophic factors bind to GDNF family receptors (GFR) and activate its co-receptor
(RET tyrosine kinase) to transduce their signals [2]. In vitro culture and slice studies reveal that
GDNF and its derivatives in the TGF family act on brain dopamine neurons and promote their development and regeneration [3-5]. Further, these neurotrophic factors enhance cell autonomous
firing and dopamine release and reuptake from their terminals [6-8], supporting their neurotrophic
role in dopaminergic development and maintenance [9]. In animal models for Parkinson’s disease,
the gene delivery of GDNF efficiently prevents midbrain dopaminergic neurons from their
neurodegeneration [10-12]. Gene knockout studies of the GDNF signal transducer RET also indicate
its neurotrophic function in the dopaminergic system [13,14].
However, various in vivo findings contraindicate the above neurotrophism of GDNF. Analyses
of GDNF-full deficient mice reveal that the no apparent decreases of dopaminergic cells, fibers, and
monoamines are detected in these mutant mice under certain genetic backgrounds or breeding 3
conditions [15]. Further, mice with heterozygous mutations in the GDNF gene rather exhibit
increased levels of dopamine and tyrosine hydroxylase (TH) [16]. A similar discrepancy associated
with GDNF neurotrophism occurs when exogenous GDNF is provided [17]. Overexpression of the
GDNF gene from a viral vector down-regulates dopaminergic neurotransmission [18-22]. In this
context, in vivo effects of GDNF hypersignaling on brain dopaminergic system remain to be
characterized.
What causes the distinct and/or discrepant actions of GDNF hypersignals on the dopaminergic
system? In the present investigation, we exmamined the neurotrophic actions of a GDNF transgene
in vivo and attempted to illustrate the in vivo role of GDNF in the nigrostriatal dopamine system.
2. Materials and methods
2.1. Animals
Mice were housed in plastic cages (200 mm × 300 mm × 140 mm) with food and water
available ad libitum. Each cage contained 2–4 mice and was kept in a temperature-controlled room
(22 ± 1 °C) under a 12–12-h light–dark cycle (8:00 on, 20:00 off). Only male mice were used for
experiments to avoid the infleunces of female estrus cycle. The animal experiments described here
were approved by the Animal Use and Care Committee of Niigata University and were performed in
accordance with the Guiding Principles for Care and Use of Laboratory Animals (NIH, USA). 4
2.2. Glial cell line-derived neurotrophic factor (GDNF) transgenic mice
A full-length mouse GDNF cDNA was inserted into the plasmid cassette pGFAP-hGH,
containing a 5′-flanking region of human glial fibrially protein gene (Gfap) and a 3′-flanking region
of the human growth hormone gene, including its polyadenylation signal sequence [23]. Prototype
transgenic mice were generated by pronuclear injection of the DNA fragment into fertilized mouse
eggs (B6/CBA strain) and were bred by crossing with wild-type mice at least five times (C57 B6/N
strain; CLEA Japan Inc., Tokyo Japan). The pure genetic background of C57B6/N transgenic mice
produced the psychobehavioral abnormality resembling Tourette syndrome or obsessive-compulsive
disorder. The mice frequently performed head grooming by themselves and produced head skin
lesion, leading to its infection. Thus, we used the sperm of the C57B6/N transgeneic mice to
inseminate the eggs of wild-type mice (FVB/N strain; CLEA Japan Inc.) to generate F1
heterozygous mice carrying both C57B6/N and FVB/N genetic backgrounds equally. These F1 mice
were genotyped using PCR with forward (5′-AGCTCACTGCAGCCTCAACCTACT-3′) and reverse
(5′-CAGGCATATTGGAGTCACTGG-3′) primers and subjected to the following experiments.
Unless the age of mice was specified, 8-12 week old mice were subjected to the following
experiments.
5
2.3. Quantification of GDNF
Mice were anesthetized using isoflurane and then decapitated. After removing the brain,
1-mm slices were prepared, and the striatum and ventral midbrain were dissected on ice. Each tissue was stored at –80 °C until use. Brain tissues were homogenized in 50 mM Tris-buffered saline
containing 1% NP-40, 1% glycerol, and protease inhibitor cocktail (Roche Diagnosis Japan, Tokyo,
Japan). Brain homogenates were centrifuged at 15000 rpm for 30 min at 4 °C, and the supernatants
were harvested. GDNF content in the supernatant was determined using an enzyme immunoassay kit
(Biogenesis, Thebarton, SA, Australia). Protein concentrations in the samples were determined using
a Micro BCA kit (Pierce, Rockland, IL, USA). The average of two measurements per sample was
normalized and used.
2.4. Immunoblotting
Brain tissues were homogenized in a 10-fold volume of lysis buffer containing 2% sodium
dodecylsulfate (SDS), 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 0.5
mM phenylarsine, and protease inhibitor cocktail (Roche Diagnosis). After centrifugation at 12500
rpm for 20 min, the supernatants were harvested, and the protein concentrations were determined
using a Micro BCA kit (Pierce Chemical, Rockland, IL). Sample buffer [5x; 0.31-M Tris-HCl (pH
6.8), 5% SDS, 50% glycerol, 25% dithiothreitol] was added to the supernatants. Samples were 6
subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane
(ADVANTEC, Tokyo, Japan) at 4 °C for 12 h. The membrane was probed with an anti-TH antibody
(1:1000; EMD Millipore, Billerica, MA), an anti-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) antibody (1:500; Santa Cruz Biotechnology, Dallas, TX). Alternatively we used the
anti-phospho Ret (Tyr 1062) antibodiy (1:1000; Santa Cruz Biotechnology, sc-20252R) and the
anti-total Ret protein antibody (1:1000; Santa Cruz Biotechnology, H-300) that have been validated
previously [24,25]. Immunoreactivity was detected using an anti-rabbit immunoglobulin antibody
conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratory, West Grove, PA)
followed by chemiluminescence detection of immune complexes (Immunostar, WAKO, Tokyo,
Japan).
2.5. Immunohistochemistry
Mice were anesthetized with chloral hydrate and perfused transcardially with 4%
paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4). Brains were removed and then fixed
in the same solution for 24 h at 4 °C. Fixed brains were immersed in 30% sucrose solution for 3–5 days and then embedded in paraffin. Sections were cut with 0.4 m thickness. After rinsing in Tris-buffered saline [TBS; 0.1 M Tris-HCl (pH 7.4), 150 mM NaCl] containing 0.2% Triton X-100,
sections were treated with 6% bovine serum albumin and 0.2% Triton X-100 in TBS and then
probed with the anti-TH antibody. After rinsing three times in TBS/0.2% Triton X-100, sections were 7
incubated with a biotinylated anti-rabbit immunoglobulin antibody. Immunoreactivity was visualized
using a Vectastin Elite ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the
substrate.
2.6. Determination of tissue content of dopamine and its metabolites
Brain tissues were homogenized in an extraction solution (0.1 M perchloric acid, 0.1 mM
EDTA). Dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid levels in the
homogenates were determined using high-performance liquid chromatography (HPLC) and
electrochemical detection as described previously [26]. Tissue pellets were homogenized in 0.5 N
NaOH, and the protein concentration was determined using Micro BCA kit (Pierce). Monoamine
contents of tissues were normalized to those of the protein concentrations.
2.7. In vivo microdialysis
Mice were anesthetized using sodium pentobarbital (65 mg/kg i.p.) and mounted on a
stereotaxic apparatus. The skull was exposed, and a hole was drilled for unilateral implantation of a
guide cannula (21-G stainless steel, 6-mm-long shaft) into the striatum (stereotaxic coordinates:
0.8-mm anterior to the bregma, 1.5-mm lateral from the midline, 1.5-mm below from the dural
surface, according to Paxions and Franklin [27]. After recovery for >10 days, microdialysis 8
experiments were performed on conscious mice. A microdialysis probe was prepared according to
Ichikawa et al. (2001) [28], and in vivo dialysis was performed according to Kato et al, [29]. Briefly,
a hollow dialysis membrane (0.31-mm o.d., 0.22-mm i.d.) (AN69HF; Hospal, Meyzieu, France) was
inserted through a 25-G stainless steel tube (0.51-mm o.d., 0.35-mm i.d., HTX-25X-12; Small parts
Inc., Miami Lake, FL, USA), and the tip was sealed with epoxy. The length of the surface exposed to
dialysis was 2 mm. A perfusate of artificial cerebrospinal fluid (147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 0.5 mM MgCl2, pH 7) was delivered at 1 L/min. During application of a depolarizing stimulus, the perfusate was exchanged for a medium containing a high concentration of potassium
(80 mM KCl, 69.7 mM NaCl, 1.2 mM CaCl2, and 0.5 mM MgCl2, pH 7).
Dopamine in the dialysates was determined using HPLC with electrochemical detection.
The mobile phase (48 mM citric acid, 24 mM sodium acetate, 10 mM NaCl, 0.5 mM EDTA, 3 mM sodium dodecyl sulfate, and 15% acetonitrile, pH 4.8) was delivered at 50 L/min [29]. Dopamine was separated using an analytical column (BDS Hypersil C18 1 × 100 mm; Thermo Fisher Scientific,
Yokohama, Japan) and detected using a 3-mm glass carbon electrode given +550 mV (Unijet flow
cell, Bioanalytical Systems Inc., West Lafayette, IN). Data analysis was performed using Epsilon LC
analysis software (Bioanalytical Systems Inc.). Data were not standardized with the recovery rate.
2.8. Analysis of locomotor activity
Exploratory motor activity was measured in a novel environment. A mouse was placed in 9
an automated activity apparatus (L × W × H: 27 cm × 27 cm × 20 cm) (MED Associates, St. Alnans,
VT) equipped with infrared photosensors at 1.62-cm intervals. We measured horizontal activity,
rearing, and stereotypy for 60 min.
2.9. Statistical analysis
To compare the differences between two groups, a two-tailed t test was used. Dopamine
concentrations in dialysates and behavioral scores were initially analyzed using repeated-measures
analysis of variance (ANOVA) with genotype as a between-subject factor and time as a
within-subject factor, followed by Tukey’s test for post-hoc comparisons. p < 0.05 indicated
statistical significance. Statistical analyzes were performed using SPSS software (SPSS, Yokohama,
Japan).
3. Results
3.1. Overexpression of GDNF protein and phosphorylation of its receptors in the transgenic mouse
brain.
To verify the expression of the introduced GDNF transgene into mice, we measured
GDNF protein content in the adult striatum and ventral midbrain, which are enriched with 10
dopaminergic terminals and cell soma, respectively. An enzyme immunoassay revealed that GDNF
tissue contents were 3–10-times higher in these regions of adult transgenic mice than in those of
wild-type littermates (Fig. 1A). To examine whether the increase in mature GDNF contents led to the
enhancement of GDNF signaling in the the transgenic mouse, we determined the phosphorylation
levels of its signaling receptor RET in the same brain regions. Immunoblotting revealed two sizes of
the immunoreactive bands for phosphorylated RET [30]. The lower size of the RET
immunoreactivity (p155) is suggested to represent the immature protein of RET found in the
intracellular membrane component and the higher size of the RET immunoreactivity (p175)
corresponds to the mature RET protein present on cell surfaces [31-33]. Accordingly, we focused on
the upper RET band and found a significant increase in the phosphorylation levels of the mature
RET protein, but no significant alterations in total RET protein levels (Fig. 1B, C). The absence of
RET down-regulation might involve the noncanonical internalization signaling and remains to be
explored further [34,35]. These results confirmed that GDNF signals were potentiated, at least, in the
major dopaminergic systems of the transgenic mice.
3.2. Influences of the GDNF transgene on phenotypic markers of dopamine neurons
To investigate the phenotypic effects of GDNF overexpression on the nigrostriatal
dopamine system, we measured protein levels of TH in the striatum and ventral midbrain (Fig. 2).
We detected a significant decrease in TH protein levels in the striatum but not in the ventral midbrain 11
(Fig. 2B, C). In agreement with the immunoblotting result, preliminary quatitative PCR indicated
that TH mRNA levels in the ventral midbrain was indistinguishable between wild and transgenic
mice (Supplemental Figure S1). We determined the time course of the TH reduction during the
postnatal development of the GDNF transgenic mice (Fig. 2C, D). On the postnatal day 3, there was
no significant difference in striatal TH levels between mouse genotypes. After postnatal day 14,
however, relative TH levels in the striatum of GDNF transgenic mice reduced to less than 80% of the
wild-type levels and continued to diminish to less than 60% at the adult stage.
To estimate the structural influences of GDNF overexpression at the adult stage, we used
TH immunostaining to assess gross anatomical features of dopaminergic fibers and cell bodies. In
agreement with the immunoblotting analysis of TH expression, the density of TH-immunoreactive
fibers were lower in the striatum of GDNF transgenic mice (Fig. 3). However, we were unable to
detect signs of neurodegeneration in these brain regions of the transgenic mice with Kluver–Barrera
staining (data not shown).
We determined dopamine metabolism in the striatum and ventral midbrain of GDNF transgenic
mice (Table 1). There were significant decreases in dopamine content and the levels of dopamine
metabolite (i.e., homovanillic acid) in the striatum. However, there were no sign of the decreases in
dopamine metabolism. Rather, dopamine content in the midbrain was elevated in the GDNF
transgenic mice (Table 1). To confirm that the decrease in dopamine content represented the
reduction in extracellular dopamine levels, we performed in vivo microdialysis of the striatum (Fig. 4
and Table 2). Basal dopamine levels were significantly lower in transgenic mice (5.3 ± 0.5 nM) than 12
in wild-type littermates (6.5 ± 0.3 nM). In contrast, basal serotonin and GABA levels were not
influenced (Table 2). To estimate the capacity of total dopamine release, we artificially evoked
dopamine release by infusing high amounts of potassium into a dialysis probe (Fig. 4). High
potassium stimulation less potentiated the extracellular levels of dopamine following high potassium
stimulation in the striata of the transgenic mice.
3.3. Effects of the GDNF transgene on behavioral traits
Striatal dopamine transmission is implicated in rodent behaviors, such as locomotor
activities [36-38]. To investigate the behavioral effects of GDNF overexpression, we measured
various locomotor scores of GDNF transgenic mice and compared with those of wild-type
littermates in a novel environment (Fig. 5). Stereotypy and rearing scores of the transgenic mice
were significantly reduced. When the whole data for holizontal travel distance were subjected to
ANOVA, we failed to detect a significant main effect of genotype with the given large variations at
0-20 min time bins. However, ANOVA for distance data at the late half time bins indicates a
significance of the genotype effect on distance, suggesting a reduction in holizontal movement of
GDNF transgenic mice only in the late time periods.
4. Discussion
13
In GNDF transgenic mice, we found significant the GDNF increase and RET phosphorylation. These
findings confirmed that the GDNF transgene was efficiently expressed in the brain regions where
dopaminergic soma and their terminals are located. Thus, we initially hypothesized that the
neurotrophic activity of mature GDNF would lead to the hyperdopaminergic phenotype associated
with the nigrostriatal dopamine pathway. However, this hypothesis was disproven by the present
analyses of TH expression and dopamine metabolism in the GDNF overexpressing transgenic mice.
The phenotypic markers of dopamine neurons, TH protein levels, TH fiber densities, tissue
dopamine content, extracellular dopamine levels, and dopamine-associated locomotor scores, all
decreased in the target brain regions of midbrain dopamine neurons in GDNF transgenic mice.
However, these phenotypes do not represent the presence of dopaminergic neurodegeneration in
these transgenic mice. Dopamine and its metabolites contents in the midbrain were rather elevated.
Our histological examination also rule out the presence of neurodegeneration in these transgenic
mice. Thus, the present in vivo findings well contrast with our current knowledge on the
neurotrophism of GDNF that have been established in culture studies [3-5]. Our findings may mirror
with the report that GDNF hetero knockout mice, which should carry low GDNF signals, display
hyperdopaminergic states in the brain [16]. With this respect, it is noteworthy that GDNF transgenic
mice with pure C57BL6 background had exhibited the psychobehavioral abnormality resembling
Tourette syndrome or obsessive-compulsive disorder (data not shown), both of which involve
dopaminergic impairments in the basal ganglia [39,40], although we cannot rule out the possibility that this behavioral abnormality might stem from transgene-induced genetic aberration. Potential 14
explanations for the neurotrophic discrepancy of these in vivo and in vitro GDNF studies are
discussed below.
In the present study, several technical limitations must be acknowledged in order to interpret the
controversial data; the sites of GDNF overexpression and cell populations targeted by exogenous
GDNF. The observed discrepancy with our initial hypothesis might be explained by the alternative
cell targets of GDNF; GABA neurons, serotonin neurons, or enteric neurons [41-44]. To test these
possibilities, we measured the extracellular levels of serotonin and GABA in the striatum but failed
to detect their changes. We also determined the protein levels of a GABAergic neuronal marker,
glutamic acid decarboxylase (GAD) as well (data not shown). However, there were no significant
alterations in these GABAergic markers. Thus, the influences of GDNF overexpression on
GABAergic and serotonergic systems appear limited, at least, in the striatum.
Bespalov et al. found that GDNF can interact with syndecan-3 to evoke a novel biological
activity of GDNF [45]. According to the finding, syndecan-3 in cortical neurons can interact to
GDNF and regulate cell migration of cortical neurons. As the potential role of this novel GDNF
receptors remain to be characterized in the present transgenic mice, we cannot rule out the
contribution of syndecan-3 to the present controversy of the GDNF action on dopamine neurons.
Similar to the present study, several previous studies report the negative effects of hyper-GDNF
signaling on dopamine synthesis and metabolism in vivo. Lentiviral vector-mediated GDNF
expression in the brain results in the down-regulation of dopamine synthesis and metabolism [18,19].
These observations contrast with the fact that the intracranial pump delivery of GDNF protein 15
induces the up-regulation of the brain dopamine system [20-22]. How can we explain this
discrepancy between the GDNF-driven dopaminergic up-regulation and down-regulation? Regarding
the site of the GDNF production, a study shows that endogenous expression of GDNF in the brain is
relatively limited to GABA neurons [46]. Unlike its name (glial cell line-derived neurotrophic factor),
the glial expression of GDNF is modest in the normal brain and only induced by brain injury [47-49].
The production site of GDNF expressed by the lentivirus vector, which down-regulated the
dopaminergic system, is an astrocyte. The site of GDNF production is same as in the present study
employing the transgenic strategy employing the glial Gfap promotor [18,19]. Therefore, we
speculate that the glial expression of GDNF might induce unknown astroglial functions or factors to
alter the biological effects of GDNF on dopamine neurons [50-51]. In agreement with this
speculation, TH levels were gradulally decreased during the postnatal period when astrocytes grow
and proliferates in the forebrain. As glial cells might also release unprocessed GDNF precursors, the
contribution of GDNF precursors also remains to be explored [52].
Although we are unable to explain the paradoxical effects of GDNF overexpression, we hope that
the present results and their interpretations will facilitate efforts to determine the inhibitory effects of
GDNF on the nigrostriatal dopamine system as well as on its action on astrocytes.
Acknowledgement
We are grateful to Ms Eiko Kitayama and Dr Hisaaki Namba for their technical assistances. We
also thank Dr Alexander Parsadanian (Department of Neurology, Washington University School of
Medicine, St. Louis) who provided us with the GDNF-overexpressing transgenic mice. 16
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22
Figure legends Fig. 1. Analysis of GDNF contents and RET phosphorylation (Y1062) in brain tissues of GDNF transgenic mice. (A) GDNF contents in the striatum (STR) and ventral midbrain (MB) were measured by ELISA (n = 5 mice each). (B) Immunoblotting for phosphorylated RET (Y1062) and total RET was performed with tissue extracts from the striatum (STR) and ventral midbrain (MB). The mark “p155” represents the immature form of RET protein (155 kDa) and the mark “p175” represents the mature form of RET protein (175kDa) [30]. (C) Phosphorylation levels of the mature RET protein (p175) were compared between transgenic mice (Tg) and wild-type littermates (Wt) (n = 4 mice each). Ratios of phosphorylated p175RET levels to GAPDH levels (mean ± SEM) were calculated and compared. *p < 0.05, **p < 0.01 by two-tailed t-test.
Fig. 2. Quantification of tyrosine hydroxylase levels in the nigrostriatal pathway. Protein lysates were prepared from the striatum (STR) and ventral midbrain (MB) of 8-week-old mice (A) as well as from the striatum of postnatal day-3 (P3), 2-week-old, and 4-week-old mice (C) and subjected to immunoblotting for tyrosine hydroxylase (TH). TH levels were statistically compared between transgenic mice (Tg) and wildtype littermates (Wt) (n = 5 mice each) (B) and among the developmental stages (D) (n = 5 mice each). Ratios of their immunoreactivities were normalized with GAPDH levels and presented with mean ± SEM. *p < 0.05, **p < 0.01 by two-tailed t-test
Fig. 3. Anatomical analysis of TH immunoreactive fibers in (A) wild types and (B) GDNF transgenic mouse. Abbreviations used: amy; amygdala; ctx; cortex, ic; internal capsule, hip; hippocampus, st; striatum, Bar: 1.0 mm
Fig. 4. Assessment of a dopamine efflux in the striatum. (A) All placements of dialysis probes in the brain were confirmed after dialysis and are shown with horizontal lines. (B) The time course of dopamine concentrations in dialysates was monitored in GDNF transgenic mice (Tg) and wildtype littermates (Wt). A back horizontal bar indicates the period when depolarizing stimulation was given by the perfusion of high concentration of potassium (80 mM KCl). Repeated measures ANOVA for a genotype x time interaction: F(6,13) = 2.335, p = 0.039. As post-hoc analysis, Tukey-Kramer comparison were performed after repeated measures ANOVA (*p < 0.05).
Fig. 5. Locomotor activities of GDNF-overexpressing transgenic mice. Spontaneous locomotor activities of GDNF-overexpressing transgenic mice (Tg) and wildtype littermates (Wt) were measured in a novel environment for 60 minutes (A) A mouse was placed in the automatic activity monitor, and horizontal movement was measured for every 5 minutes (mean ± SEM, n = 20 mice for each). (B) Stereotypic movement and (C) rearing counts were also measured simultaneously (mean ± SEM, n = 20 mice for each). Data were analyzed with repeated ANOVA with subject factors of 23
genotype and time; Distance: F(1,38) = 1.78, p = 0.19 for a gentype effect and F(11,38) = 1.65, p = 0.082 for a genotype x time interaction; Stereotypy: F(1,38) = 6.02, p = 0.019 for a genotype effect and F(11,38) = 3.75, p < 0.0001 for a genotype x time interaction; Rearing: F(1,38) = 51.0, p < 0.0001 for a genotype effect and F(11,38) = 1.46, p = 0.15 for a genotype x time interaction. post-hoc analysis of Tukey-Kramer; *p < 0.05, **p <0.01, ***p <0.001. Note; When ANOVA was applied to the late half data for distance, the genotype effect reach significance; F(1,38) = 16.78, p = 0.0002, and (**) p <0.01, (***) p <0.001 with Tukey-Kramer.
24
Table 1. Tissue contents of dopamine and its metabolites in the brain of GDNF transgenic mice ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Dopamine
DOPAC
3-MT
HVA
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Wild type
1120 + 9.7
74.3 + 1.7
72.7 + 4.4
67.2 + 2.3
GDNF transgenic
878 + 40***
70.2 + 3.8
62.9 + 2.7
57.8 + 2.2*
Wild type
38.3 + 1.3
16.6 + 0.6
ND
14.2 + 0.4
GDNF transgenic
69.9 + 4.9***
24.1 + 1.6**
ND
18.4 + 1.3**
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Data represents mean + SEM (pmol/mg protein; n = 7 each). Abbrevations used: DOPAC; 3,4-dihydroxyphenylacetic acid, 3-MT; 3-methoxytyramine, HVA; homovanillic acid, ND: not detected. *p < 0.05, **p < 0.01 and *** p < 0.001, compared to Wt littermates by unpaired two-tailed t-test.
25
Table 2. Basal levels of neurotransmitters in dialysates of the striatum ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Wild-type (nM)
Transgenic (nM)
p value
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Dopamine
6.5 ± 0.3
5.3 ± 0.5
0.044
Serotonin
3.5 ± 0.3
3.4 ± 0.3
0.92
GABA
42.6 ± 5.8
46.3 ± 8.8
0.74
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Data represent the mean ± SEM (wild-type mice, n = 7; transgenic mice, n = 8)
26
S0304-3940(17)30485-8 http://dx.doi.org/doi:10.1016/j.neulet.2017.06.005 NSL 32886
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
4-4-2017 8-6-2017 8-6-2017
Please cite this article as: Hidekazu Sotoyama, Yuriko Iwakura, Kanako Oda, Toshikuni Sasaoka, Nobuyuki Takei, Akiyoshi Kakita, Hideki Enomoto, Hiroyuki Nawa, Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sotoyama et al. NSL-17600.R2
Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor
Hidekazu Sotoyama1, Yuriko Iwakura1, Kanako Oda2, Toshikuni Sasaoka2 Nobuyuki Takei1, Akiyoshi Kakita3, Hideki Enomoto4, Hiroyuki Nawa1
1. Department of Molecular Neurobiology, Brain research institute, Niigata University 2. Department of Comparative and Experimental Medicine, Brain Research Institute,, Niigata University 3. Department of Pathology, Brain Research Institute, Niigata University 4. Laboratory for Neural Differentiation and Regeneration, Graduate School of Medicine, Kobe University
Correspondence to: Hiroyuki Nawa Department of Molecular Biology, Brain Research Institute Niigata University, Asahimachi-dori 1-757, Niigata 951-8585, Japan Tel: +81-25-227-0613, Fax: +81-25-227-0815, E-mail: [email protected]
1
HIGHLIGHTS >The expression of GDNF transgene increases RET phosphorylation in the brain. >The chronic increase in glial GDNF reduces the dopamine content and tyrosine hydroxylase activity. > Extracellular concentrations of dopamine and its metabolism decrease in the striatum of transgenic mice. > The alteration of locomotor traits of transgenic mice is consistent with dopaminergic changes.
Abstract
Glial cell line-derived neurotrophic factor (GDNF) positively regulates the development and
maintenance of in vitro dopaminergic neurons. However, the in vivo influences of GDNF signals on
the brain dopamine system are controversial and not fully defined. To address this question, we
analyzed dopaminergic phenotypes of the transgenic mice that overexpress GDNF under the control
of the glial Gfap promoter in the brain. Compared with wild-type, the GDNF transgenic mice
contained higher levels of GDNF protein and phosphorylated RET receptors in the brain. However,
there were reductions in the levels of tyrosine hydroxylase (TH), dopamine, and its metabolite
homovanillic acid in the striatum of transgenic mice. The TH reduction appeared to occur during
postnatal development. Immunohistochemistry revealed that striatal TH density was reduced in
transgenic mice with no apparent signs of neurodegeneration. In agreement with these
neurochemical traits, basal levels of extracellular dopamine and high K+-induced dopamine efflux
were decreased in the striatum of transgenic mice. We also explored the influences of GDNF
overexpression on lomomotor behavior. GDNF transgenic mice exhibited lower stereotypy and
rearing in a novel environment compared with wild-type mice. These results suggest that chronic 2
overexpression of GDNF in brain astrocytes exerts an opposing influence on nigrostriatal dopamine
metabolism and neurotransmission.
Keywords: dopamine, GDNF, RET, tyrosine hydroxylase, neurotrophic factor 1. Introduction
Glial cell line-derived neurotrophic factor (GDNF) belongs to the transforming growth
factor-beta (TGF) superfamily whose members include neurturin, artemin, persephin, and others [1]. These neurotrophic factors bind to GDNF family receptors (GFR) and activate its co-receptor
(RET tyrosine kinase) to transduce their signals [2]. In vitro culture and slice studies reveal that
GDNF and its derivatives in the TGF family act on brain dopamine neurons and promote their development and regeneration [3-5]. Further, these neurotrophic factors enhance cell autonomous
firing and dopamine release and reuptake from their terminals [6-8], supporting their neurotrophic
role in dopaminergic development and maintenance [9]. In animal models for Parkinson’s disease,
the gene delivery of GDNF efficiently prevents midbrain dopaminergic neurons from their
neurodegeneration [10-12]. Gene knockout studies of the GDNF signal transducer RET also indicate
its neurotrophic function in the dopaminergic system [13,14].
However, various in vivo findings contraindicate the above neurotrophism of GDNF. Analyses
of GDNF-full deficient mice reveal that the no apparent decreases of dopaminergic cells, fibers, and
monoamines are detected in these mutant mice under certain genetic backgrounds or breeding 3
conditions [15]. Further, mice with heterozygous mutations in the GDNF gene rather exhibit
increased levels of dopamine and tyrosine hydroxylase (TH) [16]. A similar discrepancy associated
with GDNF neurotrophism occurs when exogenous GDNF is provided [17]. Overexpression of the
GDNF gene from a viral vector down-regulates dopaminergic neurotransmission [18-22]. In this
context, in vivo effects of GDNF hypersignaling on brain dopaminergic system remain to be
characterized.
What causes the distinct and/or discrepant actions of GDNF hypersignals on the dopaminergic
system? In the present investigation, we exmamined the neurotrophic actions of a GDNF transgene
in vivo and attempted to illustrate the in vivo role of GDNF in the nigrostriatal dopamine system.
2. Materials and methods
2.1. Animals
Mice were housed in plastic cages (200 mm × 300 mm × 140 mm) with food and water
available ad libitum. Each cage contained 2–4 mice and was kept in a temperature-controlled room
(22 ± 1 °C) under a 12–12-h light–dark cycle (8:00 on, 20:00 off). Only male mice were used for
experiments to avoid the infleunces of female estrus cycle. The animal experiments described here
were approved by the Animal Use and Care Committee of Niigata University and were performed in
accordance with the Guiding Principles for Care and Use of Laboratory Animals (NIH, USA). 4
2.2. Glial cell line-derived neurotrophic factor (GDNF) transgenic mice
A full-length mouse GDNF cDNA was inserted into the plasmid cassette pGFAP-hGH,
containing a 5′-flanking region of human glial fibrially protein gene (Gfap) and a 3′-flanking region
of the human growth hormone gene, including its polyadenylation signal sequence [23]. Prototype
transgenic mice were generated by pronuclear injection of the DNA fragment into fertilized mouse
eggs (B6/CBA strain) and were bred by crossing with wild-type mice at least five times (C57 B6/N
strain; CLEA Japan Inc., Tokyo Japan). The pure genetic background of C57B6/N transgenic mice
produced the psychobehavioral abnormality resembling Tourette syndrome or obsessive-compulsive
disorder. The mice frequently performed head grooming by themselves and produced head skin
lesion, leading to its infection. Thus, we used the sperm of the C57B6/N transgeneic mice to
inseminate the eggs of wild-type mice (FVB/N strain; CLEA Japan Inc.) to generate F1
heterozygous mice carrying both C57B6/N and FVB/N genetic backgrounds equally. These F1 mice
were genotyped using PCR with forward (5′-AGCTCACTGCAGCCTCAACCTACT-3′) and reverse
(5′-CAGGCATATTGGAGTCACTGG-3′) primers and subjected to the following experiments.
Unless the age of mice was specified, 8-12 week old mice were subjected to the following
experiments.
5
2.3. Quantification of GDNF
Mice were anesthetized using isoflurane and then decapitated. After removing the brain,
1-mm slices were prepared, and the striatum and ventral midbrain were dissected on ice. Each tissue was stored at –80 °C until use. Brain tissues were homogenized in 50 mM Tris-buffered saline
containing 1% NP-40, 1% glycerol, and protease inhibitor cocktail (Roche Diagnosis Japan, Tokyo,
Japan). Brain homogenates were centrifuged at 15000 rpm for 30 min at 4 °C, and the supernatants
were harvested. GDNF content in the supernatant was determined using an enzyme immunoassay kit
(Biogenesis, Thebarton, SA, Australia). Protein concentrations in the samples were determined using
a Micro BCA kit (Pierce, Rockland, IL, USA). The average of two measurements per sample was
normalized and used.
2.4. Immunoblotting
Brain tissues were homogenized in a 10-fold volume of lysis buffer containing 2% sodium
dodecylsulfate (SDS), 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 0.5
mM phenylarsine, and protease inhibitor cocktail (Roche Diagnosis). After centrifugation at 12500
rpm for 20 min, the supernatants were harvested, and the protein concentrations were determined
using a Micro BCA kit (Pierce Chemical, Rockland, IL). Sample buffer [5x; 0.31-M Tris-HCl (pH
6.8), 5% SDS, 50% glycerol, 25% dithiothreitol] was added to the supernatants. Samples were 6
subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane
(ADVANTEC, Tokyo, Japan) at 4 °C for 12 h. The membrane was probed with an anti-TH antibody
(1:1000; EMD Millipore, Billerica, MA), an anti-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) antibody (1:500; Santa Cruz Biotechnology, Dallas, TX). Alternatively we used the
anti-phospho Ret (Tyr 1062) antibodiy (1:1000; Santa Cruz Biotechnology, sc-20252R) and the
anti-total Ret protein antibody (1:1000; Santa Cruz Biotechnology, H-300) that have been validated
previously [24,25]. Immunoreactivity was detected using an anti-rabbit immunoglobulin antibody
conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratory, West Grove, PA)
followed by chemiluminescence detection of immune complexes (Immunostar, WAKO, Tokyo,
Japan).
2.5. Immunohistochemistry
Mice were anesthetized with chloral hydrate and perfused transcardially with 4%
paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4). Brains were removed and then fixed
in the same solution for 24 h at 4 °C. Fixed brains were immersed in 30% sucrose solution for 3–5 days and then embedded in paraffin. Sections were cut with 0.4 m thickness. After rinsing in Tris-buffered saline [TBS; 0.1 M Tris-HCl (pH 7.4), 150 mM NaCl] containing 0.2% Triton X-100,
sections were treated with 6% bovine serum albumin and 0.2% Triton X-100 in TBS and then
probed with the anti-TH antibody. After rinsing three times in TBS/0.2% Triton X-100, sections were 7
incubated with a biotinylated anti-rabbit immunoglobulin antibody. Immunoreactivity was visualized
using a Vectastin Elite ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the
substrate.
2.6. Determination of tissue content of dopamine and its metabolites
Brain tissues were homogenized in an extraction solution (0.1 M perchloric acid, 0.1 mM
EDTA). Dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid levels in the
homogenates were determined using high-performance liquid chromatography (HPLC) and
electrochemical detection as described previously [26]. Tissue pellets were homogenized in 0.5 N
NaOH, and the protein concentration was determined using Micro BCA kit (Pierce). Monoamine
contents of tissues were normalized to those of the protein concentrations.
2.7. In vivo microdialysis
Mice were anesthetized using sodium pentobarbital (65 mg/kg i.p.) and mounted on a
stereotaxic apparatus. The skull was exposed, and a hole was drilled for unilateral implantation of a
guide cannula (21-G stainless steel, 6-mm-long shaft) into the striatum (stereotaxic coordinates:
0.8-mm anterior to the bregma, 1.5-mm lateral from the midline, 1.5-mm below from the dural
surface, according to Paxions and Franklin [27]. After recovery for >10 days, microdialysis 8
experiments were performed on conscious mice. A microdialysis probe was prepared according to
Ichikawa et al. (2001) [28], and in vivo dialysis was performed according to Kato et al, [29]. Briefly,
a hollow dialysis membrane (0.31-mm o.d., 0.22-mm i.d.) (AN69HF; Hospal, Meyzieu, France) was
inserted through a 25-G stainless steel tube (0.51-mm o.d., 0.35-mm i.d., HTX-25X-12; Small parts
Inc., Miami Lake, FL, USA), and the tip was sealed with epoxy. The length of the surface exposed to
dialysis was 2 mm. A perfusate of artificial cerebrospinal fluid (147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 0.5 mM MgCl2, pH 7) was delivered at 1 L/min. During application of a depolarizing stimulus, the perfusate was exchanged for a medium containing a high concentration of potassium
(80 mM KCl, 69.7 mM NaCl, 1.2 mM CaCl2, and 0.5 mM MgCl2, pH 7).
Dopamine in the dialysates was determined using HPLC with electrochemical detection.
The mobile phase (48 mM citric acid, 24 mM sodium acetate, 10 mM NaCl, 0.5 mM EDTA, 3 mM sodium dodecyl sulfate, and 15% acetonitrile, pH 4.8) was delivered at 50 L/min [29]. Dopamine was separated using an analytical column (BDS Hypersil C18 1 × 100 mm; Thermo Fisher Scientific,
Yokohama, Japan) and detected using a 3-mm glass carbon electrode given +550 mV (Unijet flow
cell, Bioanalytical Systems Inc., West Lafayette, IN). Data analysis was performed using Epsilon LC
analysis software (Bioanalytical Systems Inc.). Data were not standardized with the recovery rate.
2.8. Analysis of locomotor activity
Exploratory motor activity was measured in a novel environment. A mouse was placed in 9
an automated activity apparatus (L × W × H: 27 cm × 27 cm × 20 cm) (MED Associates, St. Alnans,
VT) equipped with infrared photosensors at 1.62-cm intervals. We measured horizontal activity,
rearing, and stereotypy for 60 min.
2.9. Statistical analysis
To compare the differences between two groups, a two-tailed t test was used. Dopamine
concentrations in dialysates and behavioral scores were initially analyzed using repeated-measures
analysis of variance (ANOVA) with genotype as a between-subject factor and time as a
within-subject factor, followed by Tukey’s test for post-hoc comparisons. p < 0.05 indicated
statistical significance. Statistical analyzes were performed using SPSS software (SPSS, Yokohama,
Japan).
3. Results
3.1. Overexpression of GDNF protein and phosphorylation of its receptors in the transgenic mouse
brain.
To verify the expression of the introduced GDNF transgene into mice, we measured
GDNF protein content in the adult striatum and ventral midbrain, which are enriched with 10
dopaminergic terminals and cell soma, respectively. An enzyme immunoassay revealed that GDNF
tissue contents were 3–10-times higher in these regions of adult transgenic mice than in those of
wild-type littermates (Fig. 1A). To examine whether the increase in mature GDNF contents led to the
enhancement of GDNF signaling in the the transgenic mouse, we determined the phosphorylation
levels of its signaling receptor RET in the same brain regions. Immunoblotting revealed two sizes of
the immunoreactive bands for phosphorylated RET [30]. The lower size of the RET
immunoreactivity (p155) is suggested to represent the immature protein of RET found in the
intracellular membrane component and the higher size of the RET immunoreactivity (p175)
corresponds to the mature RET protein present on cell surfaces [31-33]. Accordingly, we focused on
the upper RET band and found a significant increase in the phosphorylation levels of the mature
RET protein, but no significant alterations in total RET protein levels (Fig. 1B, C). The absence of
RET down-regulation might involve the noncanonical internalization signaling and remains to be
explored further [34,35]. These results confirmed that GDNF signals were potentiated, at least, in the
major dopaminergic systems of the transgenic mice.
3.2. Influences of the GDNF transgene on phenotypic markers of dopamine neurons
To investigate the phenotypic effects of GDNF overexpression on the nigrostriatal
dopamine system, we measured protein levels of TH in the striatum and ventral midbrain (Fig. 2).
We detected a significant decrease in TH protein levels in the striatum but not in the ventral midbrain 11
(Fig. 2B, C). In agreement with the immunoblotting result, preliminary quatitative PCR indicated
that TH mRNA levels in the ventral midbrain was indistinguishable between wild and transgenic
mice (Supplemental Figure S1). We determined the time course of the TH reduction during the
postnatal development of the GDNF transgenic mice (Fig. 2C, D). On the postnatal day 3, there was
no significant difference in striatal TH levels between mouse genotypes. After postnatal day 14,
however, relative TH levels in the striatum of GDNF transgenic mice reduced to less than 80% of the
wild-type levels and continued to diminish to less than 60% at the adult stage.
To estimate the structural influences of GDNF overexpression at the adult stage, we used
TH immunostaining to assess gross anatomical features of dopaminergic fibers and cell bodies. In
agreement with the immunoblotting analysis of TH expression, the density of TH-immunoreactive
fibers were lower in the striatum of GDNF transgenic mice (Fig. 3). However, we were unable to
detect signs of neurodegeneration in these brain regions of the transgenic mice with Kluver–Barrera
staining (data not shown).
We determined dopamine metabolism in the striatum and ventral midbrain of GDNF transgenic
mice (Table 1). There were significant decreases in dopamine content and the levels of dopamine
metabolite (i.e., homovanillic acid) in the striatum. However, there were no sign of the decreases in
dopamine metabolism. Rather, dopamine content in the midbrain was elevated in the GDNF
transgenic mice (Table 1). To confirm that the decrease in dopamine content represented the
reduction in extracellular dopamine levels, we performed in vivo microdialysis of the striatum (Fig. 4
and Table 2). Basal dopamine levels were significantly lower in transgenic mice (5.3 ± 0.5 nM) than 12
in wild-type littermates (6.5 ± 0.3 nM). In contrast, basal serotonin and GABA levels were not
influenced (Table 2). To estimate the capacity of total dopamine release, we artificially evoked
dopamine release by infusing high amounts of potassium into a dialysis probe (Fig. 4). High
potassium stimulation less potentiated the extracellular levels of dopamine following high potassium
stimulation in the striata of the transgenic mice.
3.3. Effects of the GDNF transgene on behavioral traits
Striatal dopamine transmission is implicated in rodent behaviors, such as locomotor
activities [36-38]. To investigate the behavioral effects of GDNF overexpression, we measured
various locomotor scores of GDNF transgenic mice and compared with those of wild-type
littermates in a novel environment (Fig. 5). Stereotypy and rearing scores of the transgenic mice
were significantly reduced. When the whole data for holizontal travel distance were subjected to
ANOVA, we failed to detect a significant main effect of genotype with the given large variations at
0-20 min time bins. However, ANOVA for distance data at the late half time bins indicates a
significance of the genotype effect on distance, suggesting a reduction in holizontal movement of
GDNF transgenic mice only in the late time periods.
4. Discussion
13
In GNDF transgenic mice, we found significant the GDNF increase and RET phosphorylation. These
findings confirmed that the GDNF transgene was efficiently expressed in the brain regions where
dopaminergic soma and their terminals are located. Thus, we initially hypothesized that the
neurotrophic activity of mature GDNF would lead to the hyperdopaminergic phenotype associated
with the nigrostriatal dopamine pathway. However, this hypothesis was disproven by the present
analyses of TH expression and dopamine metabolism in the GDNF overexpressing transgenic mice.
The phenotypic markers of dopamine neurons, TH protein levels, TH fiber densities, tissue
dopamine content, extracellular dopamine levels, and dopamine-associated locomotor scores, all
decreased in the target brain regions of midbrain dopamine neurons in GDNF transgenic mice.
However, these phenotypes do not represent the presence of dopaminergic neurodegeneration in
these transgenic mice. Dopamine and its metabolites contents in the midbrain were rather elevated.
Our histological examination also rule out the presence of neurodegeneration in these transgenic
mice. Thus, the present in vivo findings well contrast with our current knowledge on the
neurotrophism of GDNF that have been established in culture studies [3-5]. Our findings may mirror
with the report that GDNF hetero knockout mice, which should carry low GDNF signals, display
hyperdopaminergic states in the brain [16]. With this respect, it is noteworthy that GDNF transgenic
mice with pure C57BL6 background had exhibited the psychobehavioral abnormality resembling
Tourette syndrome or obsessive-compulsive disorder (data not shown), both of which involve
dopaminergic impairments in the basal ganglia [39,40], although we cannot rule out the possibility that this behavioral abnormality might stem from transgene-induced genetic aberration. Potential 14
explanations for the neurotrophic discrepancy of these in vivo and in vitro GDNF studies are
discussed below.
In the present study, several technical limitations must be acknowledged in order to interpret the
controversial data; the sites of GDNF overexpression and cell populations targeted by exogenous
GDNF. The observed discrepancy with our initial hypothesis might be explained by the alternative
cell targets of GDNF; GABA neurons, serotonin neurons, or enteric neurons [41-44]. To test these
possibilities, we measured the extracellular levels of serotonin and GABA in the striatum but failed
to detect their changes. We also determined the protein levels of a GABAergic neuronal marker,
glutamic acid decarboxylase (GAD) as well (data not shown). However, there were no significant
alterations in these GABAergic markers. Thus, the influences of GDNF overexpression on
GABAergic and serotonergic systems appear limited, at least, in the striatum.
Bespalov et al. found that GDNF can interact with syndecan-3 to evoke a novel biological
activity of GDNF [45]. According to the finding, syndecan-3 in cortical neurons can interact to
GDNF and regulate cell migration of cortical neurons. As the potential role of this novel GDNF
receptors remain to be characterized in the present transgenic mice, we cannot rule out the
contribution of syndecan-3 to the present controversy of the GDNF action on dopamine neurons.
Similar to the present study, several previous studies report the negative effects of hyper-GDNF
signaling on dopamine synthesis and metabolism in vivo. Lentiviral vector-mediated GDNF
expression in the brain results in the down-regulation of dopamine synthesis and metabolism [18,19].
These observations contrast with the fact that the intracranial pump delivery of GDNF protein 15
induces the up-regulation of the brain dopamine system [20-22]. How can we explain this
discrepancy between the GDNF-driven dopaminergic up-regulation and down-regulation? Regarding
the site of the GDNF production, a study shows that endogenous expression of GDNF in the brain is
relatively limited to GABA neurons [46]. Unlike its name (glial cell line-derived neurotrophic factor),
the glial expression of GDNF is modest in the normal brain and only induced by brain injury [47-49].
The production site of GDNF expressed by the lentivirus vector, which down-regulated the
dopaminergic system, is an astrocyte. The site of GDNF production is same as in the present study
employing the transgenic strategy employing the glial Gfap promotor [18,19]. Therefore, we
speculate that the glial expression of GDNF might induce unknown astroglial functions or factors to
alter the biological effects of GDNF on dopamine neurons [50-51]. In agreement with this
speculation, TH levels were gradulally decreased during the postnatal period when astrocytes grow
and proliferates in the forebrain. As glial cells might also release unprocessed GDNF precursors, the
contribution of GDNF precursors also remains to be explored [52].
Although we are unable to explain the paradoxical effects of GDNF overexpression, we hope that
the present results and their interpretations will facilitate efforts to determine the inhibitory effects of
GDNF on the nigrostriatal dopamine system as well as on its action on astrocytes.
Acknowledgement
We are grateful to Ms Eiko Kitayama and Dr Hisaaki Namba for their technical assistances. We
also thank Dr Alexander Parsadanian (Department of Neurology, Washington University School of
Medicine, St. Louis) who provided us with the GDNF-overexpressing transgenic mice. 16
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22
Figure legends Fig. 1. Analysis of GDNF contents and RET phosphorylation (Y1062) in brain tissues of GDNF transgenic mice. (A) GDNF contents in the striatum (STR) and ventral midbrain (MB) were measured by ELISA (n = 5 mice each). (B) Immunoblotting for phosphorylated RET (Y1062) and total RET was performed with tissue extracts from the striatum (STR) and ventral midbrain (MB). The mark “p155” represents the immature form of RET protein (155 kDa) and the mark “p175” represents the mature form of RET protein (175kDa) [30]. (C) Phosphorylation levels of the mature RET protein (p175) were compared between transgenic mice (Tg) and wild-type littermates (Wt) (n = 4 mice each). Ratios of phosphorylated p175RET levels to GAPDH levels (mean ± SEM) were calculated and compared. *p < 0.05, **p < 0.01 by two-tailed t-test.
Fig. 2. Quantification of tyrosine hydroxylase levels in the nigrostriatal pathway. Protein lysates were prepared from the striatum (STR) and ventral midbrain (MB) of 8-week-old mice (A) as well as from the striatum of postnatal day-3 (P3), 2-week-old, and 4-week-old mice (C) and subjected to immunoblotting for tyrosine hydroxylase (TH). TH levels were statistically compared between transgenic mice (Tg) and wildtype littermates (Wt) (n = 5 mice each) (B) and among the developmental stages (D) (n = 5 mice each). Ratios of their immunoreactivities were normalized with GAPDH levels and presented with mean ± SEM. *p < 0.05, **p < 0.01 by two-tailed t-test
Fig. 3. Anatomical analysis of TH immunoreactive fibers in (A) wild types and (B) GDNF transgenic mouse. Abbreviations used: amy; amygdala; ctx; cortex, ic; internal capsule, hip; hippocampus, st; striatum, Bar: 1.0 mm
Fig. 4. Assessment of a dopamine efflux in the striatum. (A) All placements of dialysis probes in the brain were confirmed after dialysis and are shown with horizontal lines. (B) The time course of dopamine concentrations in dialysates was monitored in GDNF transgenic mice (Tg) and wildtype littermates (Wt). A back horizontal bar indicates the period when depolarizing stimulation was given by the perfusion of high concentration of potassium (80 mM KCl). Repeated measures ANOVA for a genotype x time interaction: F(6,13) = 2.335, p = 0.039. As post-hoc analysis, Tukey-Kramer comparison were performed after repeated measures ANOVA (*p < 0.05).
Fig. 5. Locomotor activities of GDNF-overexpressing transgenic mice. Spontaneous locomotor activities of GDNF-overexpressing transgenic mice (Tg) and wildtype littermates (Wt) were measured in a novel environment for 60 minutes (A) A mouse was placed in the automatic activity monitor, and horizontal movement was measured for every 5 minutes (mean ± SEM, n = 20 mice for each). (B) Stereotypic movement and (C) rearing counts were also measured simultaneously (mean ± SEM, n = 20 mice for each). Data were analyzed with repeated ANOVA with subject factors of 23
genotype and time; Distance: F(1,38) = 1.78, p = 0.19 for a gentype effect and F(11,38) = 1.65, p = 0.082 for a genotype x time interaction; Stereotypy: F(1,38) = 6.02, p = 0.019 for a genotype effect and F(11,38) = 3.75, p < 0.0001 for a genotype x time interaction; Rearing: F(1,38) = 51.0, p < 0.0001 for a genotype effect and F(11,38) = 1.46, p = 0.15 for a genotype x time interaction. post-hoc analysis of Tukey-Kramer; *p < 0.05, **p <0.01, ***p <0.001. Note; When ANOVA was applied to the late half data for distance, the genotype effect reach significance; F(1,38) = 16.78, p = 0.0002, and (**) p <0.01, (***) p <0.001 with Tukey-Kramer.
24
Table 1. Tissue contents of dopamine and its metabolites in the brain of GDNF transgenic mice ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Dopamine
DOPAC
3-MT
HVA
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿
1120 + 9.7
74.3 + 1.7
72.7 + 4.4
67.2 + 2.3
GDNF transgenic
878 + 40***
70.2 + 3.8
62.9 + 2.7
57.8 + 2.2*
Wild type
38.3 + 1.3
16.6 + 0.6
ND
14.2 + 0.4
GDNF transgenic
69.9 + 4.9***
24.1 + 1.6**
ND
18.4 + 1.3**
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Data represents mean + SEM (pmol/mg protein; n = 7 each). Abbrevations used: DOPAC; 3,4-dihydroxyphenylacetic acid, 3-MT; 3-methoxytyramine, HVA; homovanillic acid, ND: not detected. *p < 0.05, **p < 0.01 and *** p < 0.001, compared to Wt littermates by unpaired two-tailed t-test.
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Table 2. Basal levels of neurotransmitters in dialysates of the striatum ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Wild-type (nM)
Transgenic (nM)
p value
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Dopamine
6.5 ± 0.3
5.3 ± 0.5
0.044
Serotonin
3.5 ± 0.3
3.4 ± 0.3
0.92
GABA
42.6 ± 5.8
46.3 ± 8.8
0.74
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿ Data represent the mean ± SEM (wild-type mice, n = 7; transgenic mice, n = 8)
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