Dopamine and α-synuclein dysfunction in Smad3 null mice.

Abstract

Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra (SN). Transforming growth factor-β1 (TGF-β1) levels increase in patients with PD, although the effects of this increment remain unclear. We have examined the mesostriatal system in adult mice deficient in Smad3, a molecule involved in the intracellular TGF-β1 signalling cascade.
Striatal monoamine oxidase (MAO)-mediated dopamine (DA) catabolism to 3,4-dihydroxyphenylacetic acid (DOPAC) is strongly increased, promoting oxidative stress that is reflected by an increase in glutathione levels. Fewer astrocytes are detected in the ventral midbrain (VM) and striatal matrix, suggesting decreased trophic support to dopaminergic neurons. The SN of these mice has dopaminergic neuronal degeneration in its rostral portion, and the pro-survival Erk1/2 signalling is diminished in nigra dopaminergic neurons, not associated with alterations to p-JNK or p-p38. Furthermore, inclusions of α-synuclein are evident in selected brain areas, both in the perikaryon (SN and paralemniscal nucleus) or neurites (motor and cingulate cortices, striatum and spinal cord). Interestingly, these α-synuclein deposits are detected with ubiquitin and P(S129)-α-synuclein in a core/halo cellular distribution, which resemble those observed in human Lewy bodies (LB).
Smad3 deficiency promotes strong catabolism of DA in the striatum (ST), decrease trophic and astrocytic support to dopaminergic neurons and may induce α-synuclein aggregation, which may be related to early parkinsonism. These data underline a role for Smad3 in α-synuclein and DA homeostasis, and suggest that modulatory molecules of this signalling pathway should be evaluated as possible neuroprotective agents.

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Dopamine and α-synuclein dysfunction in Smad3
null mice
Tapia-González et al.
Tapia-González et al. Molecular Neurodegeneration 2011, 6:72
http://www.molecularneurodegeneration.com/content/6/1/72 (13 October 2011)

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RESEARCH ARTICLE
Dopamine and a-synuclein dysfunction in Smad3
null mice
Open Access
Silvia Tapia-González1,3, Rosa M Giráldez-Pérez1,3, M Isabel Cuartero1,3, M José Casarejos1,3, M Ángeles Mena1,3,
Xiao-Fan Wang2and Amelia Sánchez-Capelo1,3*
Abstract
Background: Parkinson’s disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra
(SN). Transforming growth factor-b1 (TGF-b1) levels increase in patients with PD, although the effects of this
increment remain unclear. We have examined the mesostriatal system in adult mice deficient in Smad3, a molecule
involved in the intracellular TGF-b1 signalling cascade.
Results: Striatal monoamine oxidase (MAO)-mediated dopamine (DA) catabolism to 3,4-dihydroxyphenylacetic acid
(DOPAC) is strongly increased, promoting oxidative stress that is reflected by an increase in glutathione levels.
Fewer astrocytes are detected in the ventral midbrain (VM) and striatal matrix, suggesting decreased trophic
support to dopaminergic neurons. The SN of these mice has dopaminergic neuronal degeneration in its rostral
portion, and the pro-survival Erk1/2 signalling is diminished in nigra dopaminergic neurons, not associated with
alterations to p-JNK or p-p38. Furthermore, inclusions of a-synuclein are evident in selected brain areas, both in the
perikaryon (SN and paralemniscal nucleus) or neurites (motor and cingulate cortices, striatum and spinal cord).
Interestingly, these a-synuclein deposits are detected with ubiquitin and PS129-a-synuclein in a core/halo cellular
distribution, which resemble those observed in human Lewy bodies (LB).
Conclusions: Smad3 deficiency promotes strong catabolism of DA in the striatum (ST), decrease trophic and
astrocytic support to dopaminergic neurons and may induce a-synuclein aggregation, which may be related to
early parkinsonism. These data underline a role for Smad3 in a-synuclein and DA homeostasis, and suggest that
modulatory molecules of this signalling pathway should be evaluated as possible neuroprotective agents.
Keywords: Smad3, Parkinson’s disease, Synucleinopathy, Dopamine, Astrocytes, TGF-β, α-Synuclein, MAO-B, MAPK
Background
PD is characterized by the progressive degeneration
of dopaminergic neurons in the SN pars compacta. These
neurons project to the ST and secrete DA in order
to control voluntary movements and rewarding events [1].
Another significant feature of PD is the presence of cyto-
plasmic and neuritic inclusions of a-synuclein, known
as LB and neurites. The aetiology of idiopathic PD is
unknown, although current hypotheses focus on increased
oxidative stress, aberrant protein folding, defective protea-
some degradation or mitochondrial dysfunction. Given this
diversity in the underlying molecular mechanisms, it has
been suggested that multiple factors may cause the disease
[2]. However, the central mechanisms that induce dopami-
nergic neuron loss and a-synuclein aggregation remain
unclear.
TGF-b1 is a cytokine that might mediate some molecu-
lar mechanisms of the disease. This molecule is expressed
at low levels in uninjured brains, while it is up-regulated
in the brain in association with diseases such as Parkin-
son’s and Alzheimer’s disease, Down syndrome, ischemic
lesion, hydrocephalus or spinal cord injury [3-10].
TGF-b1 is a highly pleiotropic molecule that can regu-
late cell proliferation, migration, differentiation and apop-
tosis. Its effect is cell type and context-dependent, and it
may provide signals for both cell survival and apoptosis
[11]. Moreover, a role for TGF-b in neuronal plasticity is
becoming evident. In Drosophila, the maintenance and
* Correspondence: amelia.capelo@hrc.es
1Departamento de Neurobiología-Investigación, Hospital Ramón y Cajal,
IRYCIS, Madrid, Spain
Full list of author information is available at the end of the article
Tapia-González et al. Molecular Neurodegeneration 2011, 6:72
http://www.molecularneurodegeneration.com/content/6/1/72
© 2011 Tapia-González et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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specialization of synapses at the neuromuscular junction
requires TGF-b-mediated transcriptional regulation
[12,13]. In Aplysia, TGF-b1 induces long-term facilitation
in sensory-motor synapses [14].
During mammalian embryonic development, TGF-b3
(but not TGF-b1) is necessary for the survival of midbrain
dopaminergic neurons at perinatal stages [15]. Hence,
while TGF-b3 appears to exert its effects on newborns
neurons, TGF-b1 might have pathological effects in adults.
The context-dependent effects of these factors are clearly
observed in sensory neurons, where ontogenic neuronal
death is increased by TGF-b [16]. In the nigrostriatal sys-
tem of MPTP-treated mice, adenoviral overexpression of
active TGF-b1 produces a decrease in the survival of dopa-
minergic neurons, accompanied by higher levels of striatal
DA depletion [17,18]. Despite these studies, the role of
TGF-b in the adult mesostriatal system is not well
characterized.
The TGF-b subfamily of cytokines exert their effect by
binding to heteromeric receptor complexes at the cell sur-
face that contain a type I (primarily ALK5, but also ALK2
or ALK1) and a type II receptor (TbR-II or ActR-IIB).
Ligand binding to the receptor complex promotes the
recruitment and phosphorylation of Smad3 and/or Smad2.
Activated Smad3/2 interacts with Smad4 inducing their
translocation to the nucleus, where they bind to DNA
through specific Smad-binding elements, thereby promot-
ing targeted gene transcription. Some of the ligands that
can activate Smad3 and/or Smad2 are TGF-b1, -b2, -b3,
Inhibin, Activin, Nodal, GDF1, Vg1, and Lefty. This cano-
nical TGF-b pathway can cross-talk at multiple levels with
other signalling pathways such as those mediated by
MAPK, Fas, CaMKII, PKC, etc [11].
To address the role of TGF-b in the adult nigrostriatal
system, we have analyzed Smad3 null mice. We provide
evidence that Smad3 deficiency diminishes the trophic
and astrocytic support to nigral dopaminergic neurons, as
well as strongly altering DA metabolism. In addition a-
synuclein over-expression is produced in these mice, with
the formation of aggregates in different telencephalic,
mesencephalic and romboencephalic regions.
Results
Smad3 is expressed in dopaminergic neurons of the SN
To determine whether molecules related to TGF-b sig-
nalling are expressed in the adult murine nigrostriatal
system, we examined the expression of mRNA transcripts
encoding: TGF-b1, -b2 and -b3 ligands; type I receptors
ALK1, ALK2 and ALK5; the type II receptor TbR-II; the
Smad signalling molecules Smad2, Smad3, Smad4; and
the inhibitor Smad7. RT-PCR analysis showed that all
these molecules are expressed in the VM and ST of adult
mice, with the exception of TGF-b3 and AKL1 in the
VM (Figure 1A). The absence of TGF-b3 expression in
the VM suggests that this molecule is not critical for
Smad3 signalling in adults, in contrast with perinatal
stages [15]. We further assessed whether TGF-b signal-
ling might be present in dopaminergic neurons by histo-
logical analysis of Smad3 on tyrosine hydroxylase (TH)
positive neurons. Dopaminergic neurons in the SN
express Smad3, primarily in the cytoplasm, although
some neurons with nuclear localization were also
detected (Figure 1B, 2F, arrows). We also observed a
population of TH+/Smad3-and Smad3+/TH-cells in the
SN. The presence of Smad3 in TH-ir cells indicates that
TGF-b ligands can act directly on dopaminergic neurons.
We further assessed Smad3 expression in other brain
areas related to parkinsonism, such as the ST and the
motor cortex. Confocal analysis suggested that Smad3
was present in the nucleus of cells of the striatal matrix
compartment (the TH-fibre rich region). On the other
hand, TH-fibre innervation was detected throughout the
motor cortex, with Smad3 mainly expressed in the
nucleus of cells of the third cortical layer (Figure 1B).
Smad3 depletion leads to fewer dopaminergic neurons in
the rostral SN
Because TGF-b1 can provide both pro-survival or death
signals to cells, we determined whether Smad3 deficiency
altered the number of dopaminergic neurons in the VM.
We analyzed the nigrostriatal system in 2- to 3 -month-
old Smad3 knock-out male mice in the basal state. These
mice develop to term and live to adulthood, surviving to
2 to 4 months of age. They exhibited subtle phenotypic
abnormalities, such as osteopenia, while 1/3 of the mice
developed malformations of the forelimbs and rib cage
[19], curvature of the spine (kyphosis) and resting tremor
(data not shown). The brains of Smad3-/-mice exhibited
a grossly normal morphology. Indeed, when we used
unbiased stereological methods to quantify the number
of dopaminergic neurons along the rostro-caudal axis of
the A9, A10 and A8 areas of Smad3-/-and Smad3+/+
mice, the numbers were similar in the null and wild-type
mice genotypes in both the A10 and A8 dopaminergic
areas (Figure 2A, Additional file 1A). However, Smad3
depletion led to a 34.8% decrease in the number of TH-ir
neurons in the rostral SN (P = 0.004, Figure 2B-C). We
could not detect stronger Smad3 expression in the rostral
portion of the SN (data not shown), suggesting a func-
tional spatial differentiation of this signalling molecule.
Although the number of dopaminergic neurons
decreased in the rostral portion of SN in Smad3 null
mice, TH protein levels in the VM were similar to those
found in control mice (P = 0.566, data not shown). We
assessed apoptosis in the SN of these mice using TUNEL
assay (to measure nuclear DNA fragmentation) and mor-
phological criteria (shrunken, smooth-appearing nuclei
that have lost the granularity, associated with nuclear
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Figure 1 Smad3 is expressed in dopaminergic neurons of the SN. (A) Expression of TGFb ligands, receptors and Smad3 signalling molecules
in the VM and ST of adult mice. RT-PCR from three adult mice are shown under basal conditions for the expression of TGF-b1, -b2, and -b3
ligands, ALK1, ALK2, ALK5 type I receptors, TbR-II type II receptor, Smad2 and Smad3 receptor activated Smads, the common Smad4, and the
inhibitory Smad7. Every PCR reaction was performed using 1 μl of the cDNA pool, except for TGF-b3 and ALK1 in the VM where 1, 2 and 3 μl
were tested. (B) Coronal sections were double immunostained for TH and Smad3. SN dopaminergic neurons express Smad3 in the cytoplasm,
although some neurons have Smad3 in the nucleus (arrows). Quantitative estimate of the values representing colocalized pixels in the dual-color
image shows the co-localization of both signals in the SN (Pearson correlation coefficient = 0.62). Confocal images of TH/Smad3 double-labelling
in the ST and the motor cortex (Cx-M) suggested region co-localization, but not at the cellular level. Scale bar 50 μm.
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chromatin condensation) [20]. We could not detect a
clear increase in apoptotic cells in the SN of Smad3-/-
mice (Additional file 1B). Considering that the criteria
used to measure apoptosis last for a few hours (both
nuclear condensation and fragmentation), it seemed pos-
sible that neuronal death might be greater at older ages.
Unfortunately, these knockout mice did not survive
beyond 4 months of age and thus, an analysis of older
animals was not possible.
TGF-b3, but not TGF-b1, has been shown to be
required for the survival of dopaminergic neurons in
perinatal stages [15]. In order to discriminate between a
developmental defect and a degenerative process in the
SN of adult Smad3 null mice, we quantified the number
of rostral TH-ir neurons in day 0 of postnatal age (P0).
Similar number of dopaminergic neurons was found in
Smad3 null mice and control mice (Figure 2E), indicating
a postnatal degenerative process in Smad3 deficiency
(Figure 2C).
We evaluated whether Smad3 was necessary to maintain
dopaminergic striatal innervation from nigral dopaminer-
gic neurons, and whether the rostral deficit in DA neurons
might have a positional effect in the ST. We quantified the
optical density of TH-ir fibres along the rostro-caudal axis,
as well as in the dorsal and ventral ST. No changes were
observed between the wild type and mutant mice (Addi-
tional file 2A). Furthermore, there was no alteration in
striatal TH protein content (Additional file 2B). Dopamine
transporter (DAT) immunoreactivity was also assayed in
the ST of these mice. Smad3 deficiency resulted in a not-
statistically significant trend to decrease DAT expression
in the rostral ST (P = 0.095, Additional file 3A-C). This
trend is observed in the rostral ST, similar to the rostral
decrease on TH-ir neurons in the SN, although in a wider
Figure 2 Degeneration of dopaminergic neurons in the rostral SN of Smad3 null mice. (A) Quantification of TH-ir neurons was determined
using unbiased stereological methods in the midbrain A9, A10 and A8 dopaminergic areas (which include the SN, ventral tegmental area and
retrorubral field, respectively). No clear differences were observed between mice when the entire pool of TH-ir cells was counted in these areas.
The stereological counting method had a CE < 0.07 for each mouse. (B-C) Representation of the SN (A9) along the rostro-caudal axis showed
that Smad3-/-mutant mice had fewer TH-ir neurons than Smad3+/+in the initial rostral 500 μm of the SN (*P < 0.05, **P < 0.01, Mann-Whitney
Rank Sum test). Similar values were detected between mice in the middle and caudal regions. Unpaired t-test, n = 5 for Smad3+/+and n = 6 for
Smad3-/-mice. (D) Unbiased stereological quantification of Nissl(+) neurons in the ST of Smad3+/+and Smad3-/-mice (Unpaired t-test, n = 4
mice per genotype). CE ≤ 0.05. (E) Quantification of TH-ir neurons in the rostral portion of the midbrain at perinatal stage (P0). Similar values
were found between Smad3+/+and Smad3-/-mice, indicating no developmental defect, but a postnatal degenerative process in Smad3
deficiency (Student’s t-test, n = 3-4 per genotype). (F) Confocal microscopic images of Smad3/TH double-labelled neurons in the SN of Smad3+/+
and Smad3-/-mice. In control tissues, some neurons show Smad3 translocated to the nucleus (arrow). Smad3 null mutant mice have no
expression of the protein.
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