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A calcineurin- and NFAT-dependent pathway is
involved in a-synuclein-induced degeneration
of midbrain dopaminergic neurons
Jing Luo1,2, Lixin Sun2, Xian Lin2,
{
, Guoxiang Liu2, Jia Yu2, Loukia Parisiadou2,
Chengsong Xie2, Jinhui Ding3and Huaibin Cai2,∗
1
Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology
Beijing Key Laboratory, Beijing 100875, China,
2
Transgenics Section and and
3
Bioinformatics Core, Laboratory of
Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA
Received May 29, 2014; Revised and Accepted July 14, 2014
Parkinson’s disease (PD), the most common degenerative movement disorder, is caused by a preferential loss of
midbrain dopaminergic (mDA) neurons. Both a-synuclein (a-syn) missense and multiplication mutations have
been linked to PD. However, the underlying intracellular signalling transduction pathways of a-syn-mediated
mDA neurodegeneration remain elusive. Here, we show that transgenic expression of PD-related human
a-syn A53T missense mutation promoted calcineurin (CN) activity and the subsequent nuclear translocation
of nuclear factor of activated T cells (NFATs) in mDA neurons. a-syn enhanced the phosphatase activity of CN
in both cell-free assays and cell lines transfected with either human wild-type or A53T a-syn. Furthermore, over-
expression of a-syn A53T mutation significantly increased the CN-dependent nuclear import of NFATc3 in the
mDA neurons of transgenic mice. More importantly, a pharmacological inhibition of CN by cyclosporine A
(CsA) ameliorated the a-syn-induced loss of mDA neurons. These findings demonstrate an active involvement
of CN- and NFAT-mediated signalling pathway in a-syn-mediated degeneration of mDA neurons in PD.
INTRODUCTION
Parkinson’s disease (PD) is pathologically characterized by a
preferential loss of midbrain dopaminergic (mDA) neurons in
the substantia nigra pas compacta (SNpc) and the presence
of a-synuclein (a-syn)-containing cytoplasmic inclusions,
termed Lewy bodies and Lewy neurites (1). Both missense mu-
tation and gene multiplication in a-syn cause autosomal domin-
ant forms of familial PD (2). In addition, the a-syn gene locus
is also associated with the more common sporadic PD (3).
Together, these genetic and neuropathological studies clearly
indicate a prominent role of a-syn in the pathogenesis of PD.
A variety of in vitro and in vivo experiments have been con-
ducted to determine the underlying pathogenic mechanisms of
the a-syn-induced degeneration of mDA neurons (4–8). For
example, a-syn has been shown to interact with and affect the ac-
tivity of the enzymes phospholipase D (9), protein kinase C,
extracellular regulated kinases (10) and protein phosphatase
2A (11). In addition, a-syn binds to Ca
2+
through a novel
C-terminal domain, which affects the functional properties of
a-syn (12). However, only a few of these studies have been
carried out in SNpc DA neurons. The signalling pathways of
a-syn-mediated mDA neuron loss remain to be established.
Calcineurin (CN) is a Ca
2+
/calmodulin-dependent serine/
threonine-specific protein phosphatase enriched in neurons
(13). The nuclear factor of activated T-cell (NFAT) family of
transcription factors, including NFATc1, NFATc2, NFATc3
and NFATc4, are the main downstream targets of CN (14).
In the resting cells, NFAT proteins are hyperphosphorylated
and mainly reside in the cytoplasm. Upon activation, NFAT pro-
teins undergo rapid dephosphorylation by CN and translocate
into the nucleus, where they regulate gene transcription, in
many cases via associations with other transcription factors
(15). While the NFAT family of transcription factors was
†
Present address: Department of Anatomy, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080, China.
∗
To whom correspondence should be addressed at: Transgenics Section, Laboratory of Neurogenetics, National Institute on Aging, National Institutes
of Health, Building 35, Room 1A116, MSC 3707, 35 Convent Drive, Bethesda, MD 20892-3707, USA. Tel: +1 3014028087; Fax: +1 3014802830;
Email: caih@mail.nih.gov
Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain
in the US.
Human Molecular Genetics, 2014 1–8
doi:10.1093/hmg/ddu377
HMG Advance Access published July 28, 2014
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initially characterized in the immune system, recent studies have
highlighted the importance of this family of proteins in neurons,
where they are involved in the regulation of synaptic plasticity,
axonal growth and neuronal survival (16,17). However, the in-
volvement of CN and NFAT in the a-syn-mediated degeneration
of mDA neurons is unclear.
In our present study, we investigated whether the presence of
pathogenic a-syn affected the CN/NFAT signalling pathway in
Human Embryonic Kidney 293 (HEK293) cells transfected with
either wild-type (WT) or PD-related A53T mutant a-syn and in
mDA neurons of a-syn A53T transgenic mice (18). We found
that the overexpression of a-syn activated the CN and NFAT
pathway in cell lines and mDA neurons, whereas the inhibition
of CN/NFAT activity protected mDA neurons against
a-syn-mediated cytotoxicity.
RESULTS
a-syn activates the phosphatase activity of calcineurin
in cell-free assays
The CN phosphatase activity can be determined using the
chromogenic substrate para-nitrophenyl phosphate (p-NPP)
(19). We found that recombinant human a-syn proteins sig-
nificantly enhanced the CN-mediated dephosphorylation of
p-NPP in a cell-free assay (Fig. 1A). We then further examined
the effect of a-syn on the CN activity by using RII peptide as
the specific substrate in additional cell-free assays (20). Com-
pared with the conveniently measurable p-NPP assay, the RII
peptide assay is more sensitive and only a small amount of CN
is needed (21). The sequence of phospho-RII peptide represents
the phosphorylation site of the regulatory subunit of cAMP-
dependent protein kinase, a well characterized and more
physiological phosphopeptide substrate (20). We found that
a-syn significantly enhanced the CN-induced dephosphoryla-
tion of phospho-RII in a dose-dependent manner (Fig. 1B).
Overexpression of WT or A53T a-syn enhances
CN enzymatic activity and induces the translocation
of NFATc1 and NFATc3 in HEK293 cells
CN normally consists of one catalytic subunit of calcineurin A
(CnA) and one regulatory subunit of calcineurin B (CnB).
The phosphatase activity of CN is fully activated upon the
calcium-dependent binding of calmodulin to the CnA – CnB
complex in response to the elevation of intracellular calcium
(21). To explore the potential regulatory role of a-syn on CN,
we examined the CN phosphatase activity and the CnA expres-
sion level in HEK293 cells transiently transfected with WT or
PD-linked mutant A53T a-syn. We found that the phosphatase
activity of CN was significantly increased by 28 and 35% in
cells transfected with either WT or A53T a-syn compared
with cells transfected with empty vectors (Fig. 2A). Moreover,
the expression levels of the CnA subunit protein were not signifi-
cantly altered in the a-syn-expressing cells (Fig. 2B). These
results suggest that the overexpression of a-syn enhanced the ac-
tivity of CN without affecting its protein expression levels.
We next examined the activation of NFAT family proteins in
a-syn-expressing cells. Antibodies against four different NFAT
family members, including NFATc1, NFATc2, NFATc3 and
NFATc4, were used to measure the expression of endogenous
NFAT in the HEK293 cells. Consistent with a previous report
(22), HEK293 cells predominantly expressed NFATc1 and
NFATc3. To investigate whether the a-syn-induced activation
of CN is sufficient to trigger the nuclear translocation of
NFAT proteins, we examined the cytoplasmic and nuclear distri-
bution of NFATc1 and NFATc3 in HEK293 cells transfected
with either empty vector or a-syn expression constructs.
Forty-eight hours after transfection, the cells were harvested
for western blot analysis. The overexpression of both WT and
A53T a-syn substantially altered the cytosolic and nuclear distri-
bution of NFATc1 and NFATc3; a significant decrease in the
cytoplasmic fraction and an increase in the nuclear fraction
were observed (Fig. 2C). Accordingly, the nucleus/cytoplasm
ratios of NFATc1 and NFATc3 proteins were significantly
increased in the a-syn-transfected cells (Fig. 2D and E). More-
over, the total NFATc1 and NFATc3 levels did not differ in
the whole cell extracts prepared from a-syn and empty vector-
transfected cells (data not shown). Together, these data demon-
strate that the overexpression of a-syn may lead to the enhanced
CN activity and subsequent nuclear translocation of the NFAT
family of transcription factors in cultured cells.
Stimulation of calcium ionophore ionomycin leads to nuclear
translocation of NFATc3 in cultured mDA neurons
We investigated the expression levels of Nfatc1, Nfatc2, Nfatc3
and Nfatc4 mRNA in the SNpc DA neurons of 12-month-old
mice by sequencing the total RNA prepared from the SNpc
DA neurons isolated by laser capture microdissection. RNA
sequencing was also used to compare the expression of Nfat
family mRNA in the whole brain of 12-month-old mice. The
RNA sequencing analyses showed that Nfatc3 mRNA was pre-
dominantly expressed in the mouse SNpc DA neurons and
whole brain (Fig. 3A). To investigate the activation of CN/
NFAT pathway in mDA neurons, we treated the cultured
neurons with ionomycin and then stained them with antibodies
specific for NFATc1-c4. Ionomycin can induce the release of
calcium from the intracellular storage place (23). The mDA
neurons did not show obvious staining for NFATc1, NFATc2
and NFATc4 (data not shown). In the resting cells, NFATc3
signals were mainly detected in the cytoplasm, whereas ionomy-
cin treatment led to an almost complete nuclear translocation of
Figure 1. a-syn activates the phosphatase activity of purified calcineurin. (A–B)
The effects of recombinant a-syn proteins on the purified CN activity were deter-
mined in vitro using p-NPP (A) or RII peptide (B) as the substrate. The CN activ-
ity assayed in the absence of a-syn represented 100% activity. Data were
presented as mean +SEM (n¼3). ∗∗∗p,0.001 compared with the control
group.
2Human Molecular Genetics, 2014
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NFATc3 (Fig. 3B). Here, tyrosine hydroxylase (TH), a cytosolic
protein(24), served not onlyas a marker for dopaminergic neurons
but also as an indicator of the cytosol of these neurons (Fig. 3B).
These results suggest that NFATc3 is abundantly expressed in
mDA neurons and can be regulated by Ca
2+
stimulation.
The nuclear translocation of NFATc3 is significantly
increased in the mDA neurons of A53T a-syn
transgenic mice
To further examine whether NFATc3 is activated in the mDA
neurons of A53T a-syn transgenic mice, we examined the
expression levels of NFATc3 in the nuclear and cytoplasmic
fractions of midbrain homogenates from 1-month-old non-
transgenic (nTg) and A53T a-syn transgenic mice by western
blot analysis. The total levels of NFATc3 were not significantly
changed in the midbrain homogenates of A53T a-syn mice com-
pared with controls (Fig. 4A). In contrast, the transgenic mouse
midbrain homogenates showed a marked increase of NFATc3
immunoreactivity in the nuclear fraction and a trend towards
the decrease of NFATc3 in the cytoplasmic fraction compared
with the controls (Fig. 4B, top panel). Moreover, a significant in-
crease in the nuclear/cytoplasmic ratio of NFATc3 was found in
the mDA neurons of A53T a-syn mice (Fig. 4B, bottom panel).
To further confirm the increased nuclear translocation of
NFATc3 in the mDA neurons of A53T a-syn transgenic mice,
we checked the subcellular distribution of NFATc1, NFATc2,
Figure 2. The overexpression of WT or A53T a-syn enhances the calcineurin en-
zymatic activity and induces the translocation of NFATc1 and NFATc3 in
HEK293 cells. (A) The CN activity was measured using a synthetic peptide,
RII, as the substrate and presented in form of millimoles of phosphate
released/mg of protein/min at 308C(n¼4). Data were presented as mean +
SEM. ∗P,0.05, ∗∗P,0.01 compared with the empty vector group. (B) Repre-
sentative western blot analyses of CN expression levels in three groups (n¼4).
Data were presented as mean +SEM. (C) Western blot detection of NFATc1 and
NFATc3 in the cytoplasmic and nuclear fractions from the control vector-,
WT- and A53T a-syn-transfected HEK293 cells (n¼4). Forty-eight hours
after transfection, the cells were harvested for western blot analysis. The
b-actin (cytoplasmic) and HDAC1 (nuclear) expression levels were used as
the loading control. The histograms represent the quantification of cytoplasmic
and nuclear NFATc3 corrected by the loading control. (D,E) Bar graph
depicts the nuclear/cytoplasmic ratios of NFATc1 and NFATc3. All histograms
in D and E represent values as a percentagecompared with the control group. Data
were presented as mean+SEM. ∗P,0.05, ∗∗P,0.01.
Figure 3. Stimulation of calcium ionophore ionomycin leads to the nuclear trans-
location of NFATc3 in cultured midbrain DA neurons. (A) RNA sequencing
reveals the expression of Nfatc1, Nfatc2, Nfatc3 and Nfatc4 in SNpc DA
neurons and the whole brain of 12-month-old control mice. Two independent
SNpc and whole brain RNA samples were analysed. (B) Representative
images show co-staining of endogenous NFATc3 (green) and TH (red) in
primary mDA neurons with (+) or without ( – ) ionomycin stimulation. Scale
bar: 20 mm.
Human Molecular Genetics, 2014 3
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NFATc3 and NFATc4 in the mDA neurons of 1-month-old
A53T a-syn transgenic and littermate nTg mice by immunos-
taining. The mDA neurons of A53T a-syn transgenic mice did
not show obvious staining for NFATc1, NFATc2 and NFATc4
antibodies (data not shown). In contrast, NFATc3 staining was
detected in the mDA neurons of both nTg and A53T a-syn trans-
genic mice (Fig. 4C). While the NFATc3 signals were mainly
detected in the cytosol of nTg neurons, they were predominantly
distributed in the nuclei of mDA neurons in A53T a-syn trans-
genic mice (Fig. 4C). An additional image analysis revealed a
significant increase in the nuclear distribution of NFATc3 in
the mDA neurons of A53T a-syn transgenic mice compared
with the controls (Fig. 4D). Therefore, both western blot and im-
munocytochemistry analyses demonstrate that overexpression
of PD-related A53T a-syn leads to the nuclear translocation of
NFATc3 in mDA neurons.
Pharmacological inhibition of CN activity ameliorated
a-syn-induced mDA neuron loss in primary culture
The immunosuppressant cyclosporine A (CsA) is a specific in-
hibitor of CN (21). We found that pre-treatment with CsA
blocked the WT a-syn-induced dephosphorylation of NFATc3
in transfected HEK293 cells (Fig. 5A). We then investigated
whether CN/NFATc3 activation was involved in the a-syn-
mediated loss of TH-positive mDA neurons (18). We treated
mDA neuronal cultures from neonatal A53T a-syn and litter-
mate control pups with the CN inhibitor CsA or vehicle
(DMSO) after 5 days in vitro and then counted the numbers of
surviving TH-positive neurons 2 days after the treatment. We
observed an 47% loss of TH-positive mDA neurons in the
vehicle-treated A53T a-syn cultures compared with the controls
(Fig. 5B). In contrast, treatment with 1 mMCsA, which did not
affect the survival of control TH-positive neurons, significantly
increased the survival of TH-positive neurons in the A53T a-syn
cultures compared with the vehicle-treated ones (Fig. 5B). Fur-
thermore, we found that CsA treatment blocked the nuclear
translocation of NFATc3 in the TH-positive dopaminergic
neurons of A53T a-syn cultures (Fig. 5C). These observations
provide direct evidence that the CN/NFATc3 pathway is
involved in a-syn-induced mDA neuron loss.
DISCUSSION
In this study, we employed a new line of a-syn A53T conditional
transgenic mice to investigate the CN/NFAT signalling pathway
in mDA neurons (18). We found that a-syn promoted the CN
phosphatase activity, leading to NFATc3 nuclear import in cell
cultures and mDA neurons of transgenic mice expressing
PD-related A53T a-syn. Moreover, the pharmacological inhib-
ition of CN activity ameliorated a-syn-induced loss of mDA
neurons. These findings suggest that the CN/NFATc3 signalling
pathway may contribute to a-syn-mediated mDA neuron loss
in PD.
a-syn overexpression has been used to generate cellular
and animal models of PD. The overexpression of WT or
mutant a-syn induces cell death in dopaminergic cell lines and
primary dopaminergic neuron cultures (8,25). Transgenic mice
expressing WT or mutant a-syn show motor deficits and
Figure 4. A53T transgenic mice show significant neuron loss accompanied by NFATc3 translocation in midbrain dopaminergic neurons. (A) Western blot was used to
determine the level of A53T a-syn overexpression in the midbrain homogenate from 1-month-old A53T transgenic mice compared with littermate nTg mice using an
antiserum (C20) recognizing both human and mouse a-syn. b-actin and TH were used as the loading control. The bar graph estimates the level of a-syn overexpression
(normalized against the TH expression) in the midbrain of 1-month-old A53T mice compared with that of age-matched littermate nTg mice (n¼4 per genotype). (B)
Western blot detection of NFATc3 in total, cytoplasmic and nuclear homogenates from the midbrains of nTg and A53T mice (n¼4 per group). The expression of
b-actin (total and cytoplasmic) or nucleoporin 62 (nuclear) was used as the loading control. The histograms represent the quantification of total, cytoplasmic and
nuclear NFATc3 corrected by the loading control. Data were presented as mean+SEM. ∗∗P,0.01 compared with the nTg group. (C) Representative images of
NFATc3 (green) and TH (red) co-staining in the midbrain sections of 1-month-old nTg and A53T mice. Topro3 (blue) staining marked the nucleus. The arrowhead
points to the cytoplasmic localization of NFATc3. The asterisks label the nuclear localization of NFATc3. Scale bar: 10 mm. (D) The percentage of total cellular
NFATc3 staining of TH-positive neurons (n¼4 animals per genotype, and n¼16 neurons per animals) and the nuclear/cytoplasmic ratio of NFATc3 staining.
All histograms represent A53T mice values as a percentage compared with the nTg group. Data were presented as mean +SEM. ∗∗P,0.01.
4Human Molecular Genetics, 2014
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changes in dopamine levels (5). Although the excessive aggrega-
tion of a-syn has been associated with neurodegeneration, the
mechanism by which a-syn injures dopaminergic neurons
remains to be fully established. Several hypotheses have been
proposed, including a-syn-induced Ca
2+
dyshomeostasis.
More recently, oligomeric forms of a-syn have been proposed
to be the most neurotoxic form of this protein. a-syn oligomers
trigger Ca
2+
influx and the subsequent caspase activation in cul-
tured neurons and neuroblastoma cells (26,27). a-syn has been
suggested to be able to form Ca
2+
permeable pores in the
plasma membrane, much like other aggregating proteins, such
as amyloid bpeptides and prion proteins (28). Notably, the
SNpc DA neurons are pace-making neurons that keep firing
through an L-type calcium channel (29). As the result, the alter-
ation of calcium homeostasis may make the SNpc DA neurons
more vulnerable to PD-related degeneration (29). In line with
this notion, the analyses of central nervous system tissues from
patients with PD suggest a role for cellular Ca
2+
overload in
the death of vulnerable neurons in this disease (29). The hypoth-
esis that a-syn may trigger Ca
2+
influx, together with the more
general concept that perturbed Ca
2+
homeostasis is of central
importance to neurodegenerative processes (30), prompted us
to determine the potential effects of increased a-syn levels on
processes downstream of the Ca
2+
-signalling pathway.
In our study, we identified a new calcium-dependent pathway
in dopaminergic neuron loss. We found that a-syn directly acti-
vated CN activity on the small phosphorylated compound p-NPP
as well as the 19 amino acid phosphopeptide RII. In addition, we
also observed a similar increase in the CN activity in WT or
A53T a-syn-transfected HEK293 cells. The activation of CN
leads to the dephosphorylation of key signal transduction mole-
cules, including the NFAT family of transcription factors (31).
The dephosphorylated NFAT is transferred from the cytosol
into the nucleus, where it induces the expression of target
genes, such as cytokine genes, in human T cells in cooperation
with other transcription factors, such as AP-1 (32). Here, we
found a typical nuclear translocation of NFATc3 in mDA
neurons in response to ionomycin stimulation. More import-
antly, we observed a significant translocation of NFATc3 from
the cytosol to the nucleus in the mDA neurons of A53T a-syn
transgenic mice. However, the function and downstream
targets of NFAT in mDA neurons remain to be determined.
Although originally described in T cells, NFATs are now
known to participate in the regulation of CN-mediated transcrip-
tional activity in axonal growth, dendritic branching and pre-
synaptic differentiation (17,33,34). A combined deletion of
either NFATc3/NFATc4 or NFATc2/NFATc3/NFATc4 iso-
forms leads to a marked deficiency in axonal development
(16). Moreover, NFATc3 and NFATc4 have also been impli-
cated in the regulation of neuronal survival (35). For example,
NFATc4 activation has also been recently proposed to mediate
deafferentation-induced neuronal loss in the cochlear nucleus
(36). Furthermore, increased CN levels and the associated shut-
tling of NFATc3 and NFATc4 from the cytosol to the nucleus are
indicated in methamphetamine-induced neuron death (37).
NFAT translocation also induced FasL protein expression in stri-
atal GABAergic neurons, which may be related to neuronal
apoptosis and cognitive defects in patients who abuse metham-
phetamine (38). Given that NFAT activation contributes to
the loss of neurons, the a-syn-mediated translocation of
NFATc3 may contribute to the mDA neurodegeneration in
PD. In support of this hypothesis, we found that treatment with
the CN inhibitor CsA rescued the a-syn-induced loss of
primary mDA neuron cultures. Notably, CsA not only inhibits
CN-dependent NFAT transcriptional activation but also blocks
the mitochondrial Ca
2+
fluxes by binding to the mitochondrial
receptor cyclophylin D (CypD) (39,40). However, whether
the mitochondrial calcium homeostasis is altered in the
mDA neurons of A53T a-syn transgenic mice remains to be
determined. In addition, it will be interesting to identify the
downstream targets of NFATc3 in mDA neurons, which
may provide new molecular targets for potential therapeutic
interventions.
MATERIALS AND METHODS
Cell line culture and transfection
HEK293 cells were cultured in 100-mm dishes with Dulbecco’s
Modified Eagle’s Medium supplemented with 10% heat-
inactivated foetal bovine serum (FBS) (Invitrogen) and
penicillin/streptomycin (Sigma – Aldrich). Six micrograms of
Figure 5. CsA treatment ameliorated A53T a-syn-induced loss of midbrain
dopaminergic neurons. (A) CsA induces NFATc3 phosphorylation in HEK293
cells transfected with WT a-syn. (B) Survival of TH-positive neurons treated
with DMSO or 1 mMCsA. Y-axis represents the survival rate (%) of TH-positive
neurons. Six pairs of control and A53T cultures were analysed for DMSO or CsA
treatment, respectively. For each culture, 200 – 500 TH-positive neurons were
counted. Data were presented as mean +SEM. ∗∗P,0.01, ∗∗∗P,0.001 com-
pared with the A53T DMSO group. (C) Representative images show co-staining
of NFATc3 (green) and TH (red) in primary mDA neurons from neonatal A53T
a-syn pups treated with CsA or DMSO. Scale bar: 20 mm.
Human Molecular Genetics, 2014 5
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WT and A53T
a
-syn cDNAs in the pcDNA3.1expression vector
(Invitrogen) were used for each transfection via the Fugene 6
Transfection Reagent (Roche Applied Science) according to
the manufacturer’s instructions. The cells were allowed to
grow for 48 h after transfection before being harvested for the
following experiments. The cell suspension was washed with
three volumes of ice-cold phosphate-buffered saline by repeated
centrifugation at 500×gfor 2 min at 48C. The cells were lysed in
appropriate buffers to determine the CN enzyme activity and for
the western blot analysis.
Nuclear and cytoplasmic fractionation
The NE-PER nuclear and cytoplasmic extraction kit (Thermo
Fisher Scientific, Inc.) was used to separate the cytoplasmic
and nuclear fractions of HEK293 cells and mouse midbrain
tissues according to the instruction of the manufacturer.
b-actin was used as the loading control for the cytosolic proteins,
and histone deacetylase-1 (HDAC1) was used as the loading
control of nuclear proteins.
Western blot analysis
The HEK293 cells or midbrain tissues were homogenized in
SDS buffer (50 mMTris – HCl, 150 mMNaCl, 2 mMEDTA, pH
7.6, and 2% SDS) supplemented with protease inhibitors
(Roche Applied). Following 15-min incubation on ice, the
protein extracts were clarified by centrifugation at 15 000 ×g
for 30 min at 48C. The protein contents of the supernatants
were quantified using an assay kit based on bicinchoninic
acid (Thermo Scientific), and the supernatants were then sepa-
rated by 4– 12% NuPage Bis-Tris –polyacrylamide gel electro-
phoresis (Invitrogen) using MES or MOPS running buffer
(Invitrogen). After transferring to nitrocellulose membranes,
the membranes were immunoblotted with the appropriate dilu-
tions of primary antibodies: a-syn (1 : 500, Santa Cruz),
b-actin (1 : 1000, Sigma), tyrosine hydroxylase (anti-TH anti-
body, 1 : 1000, Santa Cruz), NFATc3 (F-1 monoclonal antibody,
1 : 500, Santa Cruz), NFATc1 (7A6 monoclonal antibody, 1 :
500, Santa Cruz) or calcineurin (pan-calcineurin A antibody,
1 : 1000, Cell Signaling). The signals were visualized by
enhanced chemiluminescence development (Thermo Scientific)
and quantified by a Scion Image System (Frederick, MD).
Calcineurin activity assay
The CnA and CnB subunits for the activity assay were expressed
and purified according to the description (40). The protein purity
was analysed by SDS– PAGE. The purified CnA was concen-
trated with an Amicon Ultra Filter Unit and diluted in 50 mM
Tris– HCl, 0.5 mMdithiothreitol, 0.1 mg/ml BSA and 50% gly-
cerol. A colorimetric assay was used to determine the activities
of CN with 20 mMp-NPP or RII peptide as the substrate. The
reaction was terminated after reacting at 308C for 10 min.
The same vehicle without recombinant human a-syn (ProSpec,
Israel) wasused as a control. Therecombinant humana-synuclein
produced in Escherichia Coli is purified by proprietary chromato-
graphic techniques, and the purity is .95.0% as determined by
SDS– PAGE. The CN activity of each sample was determined
in triplicate. The phosphatase activities are presented as % of
the control.
The CN activity in HEK293 cells was determined with a Cal-
cineurin Cellular Assay Kit (PLUS-AK-816, Enzo Life
Sciences) according to manufacturer’s instructions. Briefly,
the CN activity was measured as the dephosphorylation rate of
the RII peptide. The amount of PO
4
released was calorimetri-
cally determined with the classic malachite green reagent. The
activity was calculated as the difference in protein phosphatase
activity in 2×assay buffer and 2 ×EGTA buffer. The CN activ-
ity of each sample was determined in triplicate. The phosphatase
activities are presented as millimoles of phosphate released/mg
of protein/min at 308C.
Human A53T
a
-syn transgenic mice
The PITX3-IRES-tTA/tetO-A53T double transgenic mice were
generated as previously described (18). Mice were housed in a
12-h light/dark cycle and fed a regular diet ad libitum. All
mouse work followed the guidelines approved by the Institution-
al Animal Care and Use Committees of the National Institute of
Child Health and Human Development, NIH.
Genotyping
The genomic DNA was extracted from a tail biopsy using the
DirectPCR Lysis Reagent (Viagen Biotech, Inc., Los Angeles,
CA, USA) and subjected to PCR amplification using specific
sets of PCR primers for each genotype, including PITX3-
IRES2-tTA transgenic mice (PITX3-F: GACTGGCTTGCC
CTCGTCCCA and PITX3-R: GTGCACCGAGGCCCCAGA
TCA), a-syn A53T transgenic mice (PrpEx2-F: TACTGCTC
CATTTTGCGTGA and SNCA-R: TCCAGAATTCCTTCCTG
TGG) and tetO-H2Bj-GFP mice (H2BGFP0872F, AAGTTC
ATCTGCACCACCG and H2BGFP1416R, TCCTTGAAGAA
GATGGTGCG).
Immunohistochemistry and light microscopy
To immune-stain the mouse midbrain sections, the mice were
sacrificed and then perfused via cardiac infusion with 4% paraf-
ormaldehyde in cold PBS. To obtain frozen sections, the brain
tissues were removed, cryo-protected in 30% sucrose for 24 h
and sectioned at 40 mm thickness using a cryostat (Leica
CM1950). Antibodies specific to NFATc3 (1 : 300, Sigma –
Aldrich USA, St. Louis, MO, USA), a-syn and tyrosine hydro-
xylase (TH) (1 : 1000, Pel-Freez Biologicals, Rogers, AR,
USA) were used as suggested by the manufacturers. Alexa
488- or Alexa 568-conjugated secondary antibody (1 : 500, Invi-
trogen) was used to visualize the staining. The fluorescence
images were captured using a laser scanning confocal micro-
scope (LSM 510; Zeiss, Thornwood, NJ, USA). Sections from
Bregma-2.92 to -3.16 mm of control and A53T transgenic
mice were used for immunostaining and the following image
analyses. Sixteen TH-positive neurons were selected from
each brain that showed intact nuclear structure based on
Topro3 staining. The paired images in all figures were collected
at the same gain and offset settings. Post-collection processing
was uniformly applied to all paired images. The images are pre-
sented as either a single optic layer after acquisition in z-series
stack scans at 0.8-mm intervals from individual fields or as
maximum intensity projections to represent confocal stacks.
6Human Molecular Genetics, 2014
at Galter Health Sciences Library on September 11, 2014http://hmg.oxfordjournals.org/Downloaded from
Laser capture microdissection and RNA-sequencing
analysis
We adopted whole-genome gene expression analyses of DA
neurons isolated from the SNpc of 12-month-old a-syn A53T
transgenic mice and age-matched control mice. To facilitate
the identification of mDA neurons, we generated the
Pitx3-tTA::tetO-H2Bj-GFP::tetO-A53T triple transgenic mice
and control Pitx3-tTA::tetO-H2Bj-GFP double transgenic
mice, in which the histone-GFP fusion proteins (H2Bj-GFP)
are restricted to the nucleus of TH-positive DA neurons in
both the SNpc and ventral tegmental area (VTA) (18). The mid-
brain DA neurons were isolated directly without any staining
owing to the strong GFP signals, and the integrity of RNA was
preserved for the later RNAseq experiments. The strong GFP
signals allowed directly isolate the midbrain DA neurons
without any staining and help to preserve the integrity of RNA
for the later RNAseq experiments. The total RNA was extracted
with the PicoPure Isolation kit (Applied Biosystems). The
genomic DNA was eliminated during the RNA isolation
process. The RNA quantity was measured with NanoDrop-
Spectrophotometer, and the quality was evaluated with BioAna-
lyzer. The libraries for TruSequencing were set up from 100-ng
total RNA fragmented to 200-base pair length. The cDNA li-
braries were amplified by PCR and validated by the BioAnaly-
zer. The deep RNA sequencing was performed on the Illumina
HiSeq 2500 on 2 ×100 bp type for 200 cycles with the Illumina
TruSeq SBS kit. After sequencing instrument generates the se-
quencing images, both image analysis step and base call steps
were run using standard Illumina pipeline. Raw sequences
were filtered and trimmed based upon quality scores over read
cycles. Then, we aligned the paired-end sequencing reads to
mouse reference genome (mm10) using Bowtie2 (2.1.0)
package and Samtools (0.1.14) toolkit. We then utilized Cuf-
flinks (2.1.1) to annotate sequencing reads and estimate tran-
scripts abundances. The 10-mm transcript sequences from
NCBI Reference Sequence Database were used as the annotation
reference. We used DEGSeq, a Bioconductor R package, in
downstream count-based analysis for differential expression
among samples with different genotypes.
Primary neuronal culture and treatment
Primary midbrain neuronal cultures were prepared from P0 pups
of breeding pairs fed with doxycycline (DOX). Briefly, individ-
ual midbrain containing SNpc and VTA was subjected to papain
digestion (5 U/ml, Worthington) for 40 min at 378C. The
digested tissue was carefully triturated into single cells using in-
creasingly smaller pipette tips. The cells were then centrifuged at
250×gfor 5 min and re-suspended in warm Basal Medium
Eagle supplemented with 5% heat-inactivated FBS, 1×B27
(Gibco), 1×N2 (Gibco), 1×GlutaMAX, 0.45% D-glucose
(Sigma), 10 U/ml penicillin (Gibco) and 10 g/ml streptomycin
(Gibco). The dissociated cells from each midbrain were
equally divided and plated onto four 12-mm round coverslips
pre-coated with poly-D-lysine and laminin (BD Bioscience),
and the slips were maintained at 378C in a 95% O
2
- and 5% CO
2
-
humidified incubator. Twenty-four hours after seeding, the
cultures were switched to serum-free medium supplemented
with 5 Mcytosine-D-arabino-furanoside (Sigma), which was
used to suppress the proliferation of glia. The cells in two
sister coverslips were maintained in the presence of 1 mg/ml
DOX after plating. After 5 days in vitro, DOX-treated or non-
treated cells were exposed to 1 mMCsA (Sigma) or DMSO
vehicle control for another 48 h. The cells were then fixed with
4% paraformaldehyde and 4% sucrose in PBS for 15 min, per-
meabilized by 0.1% Triton X-100 for 5 min and blocked in
10% non-immune donkey serum for 1 h at room temperature
(RT). The cells were then double-labelled with primary anti-
bodies against TH (1 : 1000, Santa Cruz) and NFATc3 (1 :
1000, Sigma) overnight at 48C in a humidified chamber. After
three washes with PBS, donkey-derived secondary antibodies
conjugated to Alexa Fluor 488 and Alexa Fluor 546 (1 : 1000,
Invitrogen) were applied and incubated for 1 h at RT in the
dark. After extensive washes, the nuclei were stained with
Topro3 iodide (1 : 1000, Invitrogen). Finally, the coverslips
were mounted on glass slides with Prolong Gold antifade
reagent (Invitrogen), and the fluorescence signals were detected
using a laser scanning confocal microscope (LSM 510; Zeiss).
The total number of all TH-positive neurons on each of the
four sister coverslips was counted under a 25×objective.
Owing to the different amounts of midbrain neurons in each
mouse, we used the relative survival rate of TH-positive
neurons in each midbrain. The rate was calculated by dividing
the number of TH-positive neurons on the non-DOX-treatment
coverslip with the number of TH-positive neurons on the DOX-
treated coverslip from the same midbrain preparation.
Image analysis
To quantitatively assess the marker protein distributions, images
were taken using identical settings and exported to ImageJ (NIH)
for imaging analyses. The images were converted to an 8-bit
colour scale (fluorescence intensity from 0 to 255) using
ImageJ. The areas of interest were first selected with Polygon
or Freehand selection tools and then subjected to measurement
by mean optical intensities or area fractions. The mean intensity
for the background area was subtracted from the selected area to
determine the net mean intensity.
Statistical analysis
A statistical analysis was performed using GraphPad Prism 5
(GraphPad Software, Inc., La Jolla, CA). The data are presented
as the mean +SEM. Statistical significances were determined
by comparing means of different groups and conditions
using t-test, one-way ANOVA with post hoc test. ∗P,0.05,
∗∗P,0.01, ∗∗∗P,0.001.
ACKNOWLEDGEMENTS
The authors thank members of Cai lab for providing various
supports, Mr Christopher Letson and Dr J. Raphael Gibbs for
helping with RNAseq experiments, and China Scholarship
Council (CSC) for its international exchange programs.
Conflict of Interest statement. None declared.
FUNDING
This work was supported in part by the intramural research pro-
grams of National Institute on Aging (AG000928, AG000929)
Human Molecular Genetics, 2014 7
at Galter Health Sciences Library on September 11, 2014http://hmg.oxfordjournals.org/Downloaded from
and by the National Natural Science Foundation of China
(Project 81072648 and 81373389).
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