 |
 Previous Article | Next Article 
The Journal of Neuroscience, April 15, 2002, 22(8):3090-3099
A Role for  -Synuclein in the Regulation of Dopamine
Biosynthesis
Ruth G.
Perez1,
Jack C.
Waymire3,
Eva
Lin1,
Jen J.
Liu1,
Fengli
Guo2, and
Michael J.
Zigmond1
1 Department of Neurology and 2 Center for
Biological Imaging, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15213, and 3 Department of
Neurobiology and Anatomy, University of Texas Medical School, Houston,
Texas 77030
 |
ABSTRACT |
The  -synuclein gene is implicated in the pathogenesis of
Parkinson's disease. Although -synuclein function is uncertain, the
protein has homology to the chaperone molecule 14-3-3. In addition,
-synuclein can bind to 14-3-3, and both -synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and
activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine
(DA) biosynthesis, we explored whether -synuclein also bound to
tyrosine hydroxylase and influenced its activity. Immunoprecipitation
revealed an interaction between -synuclein and tyrosine hydroxylase
in brain homogenates and MN9D dopaminergic cells. Colocalization of
-synuclein with tyrosine hydroxylase was confirmed by immunoelectron
microscopy. To explore the consequences of the interaction, we measured
the effect of recombinant -synuclein on tyrosine hydroxylase
activity in a cell-free system and observed a dose-dependent inhibition
of tyrosine hydroxylase by -synuclein. To measure the impact of
-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably
transfected MN9D cells with wild-type or A53T mutant -synuclein.
Overexpression of wild-type or A53T mutant -synuclein did not
significantly alter tyrosine hydroxylase protein levels in our stably
transfected cells. However, overexpressing cell lines had significantly
reduced tyrosine hydroxylase activity and a corresponding reduction in
dopamine synthesis. The reduction in cellular dopamine levels was not
caused by increased dopamine catabolism or dopamine efflux. These data
suggest that -synuclein plays a role in the regulation of dopamine
biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If
so, a loss of soluble -synuclein, by reduced expression or
aggregation, could increase dopamine synthesis with an accompanying
increase in reactive dopamine metabolites.
Key words:
14-3-3; MN9D; Parkinson's disease; phosphorylation; rat
brain; tyrosine hydroxylase
| |
INTRODUCTION |
Parkinson's disease (PD) is a
neurodegenerative disorder that results from the loss of dopamine
(DA)-containing neurons projecting from the substantia nigra to the
dorsal striatum (Bernheimer et al., 1973 ; Hornykiewicz and Kish, 1986).
Although PD etiology remains unknown, evidence points to a role for
-synuclein, a presynaptic protein of undetermined function (Takeda
et al., 1998; Galvin et al., 2001). Familial PD can result from
-synuclein gene mutations, producing A53T (Polymeropoulos et al.,
1997) or A30P (Kruger et al., 1998) amino acid changes. Reduced
-synuclein expression is reported in sporadic PD (Neystat et al.,
1999), and -synuclein is a major component in Lewy bodies, a
neuropathological hallmark of PD (Spillantini et al., 1997, 1998;
Jenner and Olanow, 1998; Markopoulou et al., 1999). -Synuclein
inclusions occur in several animal models (Betarbet et al., 2000; Feany
and Bender, 2000; Kowall et al., 2000; Masliah et al., 2000), and
cell-free studies confirm that -synuclein mutations accelerate
oligomerization/aggregation (Conway et al., 1998, 2000; Hashimoto et
al., 1998; Giasson et al., 1999; Narhi et al., 1999). Moreover, during
PD pathogenesis, mutant -synuclein aggregation appears to be
accelerated, because Lewy body pathology is reported in early-onset
familial PD (Golbe et al., 1996). Cumulatively, these findings
implicate -synuclein aggregation in PD pathogenesis.
-Synuclein may be a chaperone protein based on its ability to
inhibit thermally induced protein precipitation (Kim et al., 2000;
Souza et al., 2000), a hypothesis that is further supported by the
finding that -synuclein has structural homology with and binds to
14-3-3 proteins, a family of molecular chaperones (Ostrerova et al.,
1999). The homologous domains of -synuclein and 14-3-3 contain
residues that for 14-3-3 mediate protein-binding interactions (Zhang et
al., 1997). Thus, identifying proteins to which -synuclein binds may
help elucidate -synuclein function.
Early studies revealed that 14-3-3 binds to and activates tyrosine
hydroxylase (TH) (Ichimura et al., 1988), the rate-limiting enzyme in
catecholamine synthesis. 14-3-3 preferentially binds to phosphoserines
(for review, see Muslin and Xing, 2000), and short-term TH activity is
regulated by serine phosphorylation (Kumer and Vrana, 1996; Zhang et
al., 1997). For example,
Ca2+/calmodulin-dependent protein kinase
II, which is linked to depolarization-dependent Ca2+ influx, phosphorylates TH on Ser19
and Ser40 (Haycock, 1990; Kumer and Vrana, 1996). Furthermore, 14-3-3 interacts with TH through an association with Ser19 and Ser40 (Kleppe
et al., 2001), and the interaction of 14-3-3 with TH Ser19 is required
for TH activation (Itagaki et al., 1999). Because 14-3-3 and
-synuclein may affect the activities of proteins to which they both
bind (Jenco et al., 1998; Ostrerova et al., 1999; Van Der Hoeven et al., 2000), we hypothesized that -synuclein, like 14-3-3, may also
bind to TH but, in contrast, inhibit its activity. Although the
function of -synuclein remains obscure, we now provide data suggesting a role for -synuclein as a key regulator of DA synthesis. This finding suggests that a loss of -synuclein by its aggregation or its decreased expression, as occurs in PD (Neystat et al., 1999),
may selectively disrupt DA homeostasis and negatively impact DA
neuronal survival.
| |
MATERIALS AND METHODS |
Unless stated otherwise, all reagents used were obtained from
Sigma/Aldrich (St. Louis, MO).
Cell culture
The MN9D dopaminergic line is a fusion of rostral mesencephalic
neurons from embryonic C57BL/6J (embryonic day 14) mice with the N18TG2
neuroblastoma cells (gift from Drs. A. Heller and L. Wong,
University of Chicago). These cells express both TH and aromatic amino
acid decarboxylase (AADC) (Choi et al., 1991) and produce measurable
levels of DA (Choi et al., 1992). Cells were grown on Primaria tissue
culture plates (BD-Falcon Biosciences, Lexington, TN) in DMEM (D5648;
Sigma), pH 7.2, with 50 U/ml penicillin, 50 µg/ml streptomycin, and
10% fetal bovine serum (Hyclone, Logan, UT) and incubated at 37°C in
a 5% CO2 environment. Frozen stocks of low-pass
cells (<10) were used to ensure high-level transgene expression. To
provide large enough pools of cells for biochemical measurements, we
used undifferentiated MN9D cells at 60-80% confluence. To control for
clonal variability, we made biclonal pools of wild-type (WT; clones D7
and D10) and A53T mutant -synuclein (clones A7 and C8) cells by 1:1
platings using equal numbers of cells at the time of plating for each experiment.
Transfection
MN9D cells were stably transfected using pcDNA3.1 constructs for
expression of green fluorescent protein (GFP) (gift from Dr. Guodong
Cao, University of Pittsburgh), human wild-type -synuclein, or A53T
-synuclein (gifts from Dr. Yong-Jian Liu, University of Pittsburgh)
by CaPO4 precipitation (Perez et al., 1996) with minor modifications (Brunet et al., 1999). Cells in 60 mm Primaria tissue culture plates (BD-Falcon Biosciences) were at 60% confluence when transfected. Polyclonal pools of stably transfected cells and
monoclonal lines were selected with 400 µg/ml G418 and maintained in
200 µg/ml G418 added to media.
Cell viability assay
MN9D cells were plated 5000 cells per well on 96-well plates and
evaluated at 5 hr after plating and at 24 hr after switching cells to
serum-free medium. For sodium
3,3'-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}bis-4-methoxy-6-nitrobenzene sulfonic acid (XTT) assay, media were supplemented with 20 µl/well XTT (Procheck, Intergen, Purchase, NY) 1-2 hr before
spectrophotometric evaluation. Conversion of XTT to formazan was
measured at 490 nm by microplate spectrophotometry (SpectraMax 340, Molecular Devices, Sunnyvale, CA). Total protein was assessed by
bicinchoninic acid (BCA) spectrophotometry (see below) for normalizing data.
Co-immunoprecipitation
For co-immunoprecipitation (co-IP), all steps were performed at
4°C. Adult rat striata were collected, weighed, and homogenized in 5 vol of ice-cold co-IP buffer that contained 50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.3% Triton
X-100, 10% glycerol plus aprotinin, leupeptin,
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF),
 -glycerophosphate, and dithiothreitol to inhibit protease and
phosphatase activities. High stringency co-IP buffers were identical
but also contained 500 mM NaCl and 0.1% SDS. Supernatants were collected after centrifugation at 17,000 × g
(Sorvall RC5B; Kendro Laboratory Products, Newtown, CT). A control
aliquot of each supernatant was separated and frozen before co-IP for
total protein determinations. Samples were precleared for 1 hr with 10 µl 1% BSA plus 25 µl each of protein A and protein G Sepharose beads (Zymed Laboratories, South San Francisco, CA). Primary antibodies included mouse anti-14-3-3 (Transduction Laboratories, Lexington, KY),
mouse anti-TH (MAB 318, Chemicon, Temecula, CA), and mouse anti--synuclein (Syn-1, Transduction Laboratories).
Immunoprecipitating antibodies (5 µg) were coupled to SiezeX beads
according to the manufacturer (Pierce, Rockford, IL). Equal aliquots of
homogenates (5.0 mg/ml total protein) were incubated with antibodies
against -synuclein, TH, or 14-3-3, or with preabsorbed antibodies
against -synuclein or TH. Immune complexes were eluted, separated on 12-15% Tris-glycine SDS-PAGE gels, transferred to nitrocellulose, reacted with the same primary antibodies described above, and visualized by chemiluminescence. We noted elution of IgG heavy and
light chains along with antigens in some experiments. A 45 kDa band of
uncertain origin has been reported on immunoblots using the Syn-1
antibody (Payton et al., 2001). MN9D cell extracts were prepared using
the same buffer and co-IP conditions described above, and final
supernatants containing ~500 µg/ml total protein per cell line were
separated into equal volume samples for IP.
Assays for DA, dihydroxphenylalanine, dihydroxyphenylacetic
acid, and homovanillic acid
Assays were performed by HPLC with electrochemical detection as
described by Wagner et al. (1979) with minor modifications (Jackson et
al., 1993). Briefly, 20-50 µl supernatant samples were injected onto
a Symmetry C18 column (3 mm particle size; 3.9 × 150 mm; Waters
Symmetry, Milford, MA). The mobile phase consisted of 50 mM
H2NaO4P·H2O,
0.72 mM sodium octyl sulfate, 0.075 mM
Na2EDTA, and 16% methanol (v/v), pH 2.7. The
mobile phase was pumped through the system at 1.2 ml/min using a
Shimadzu LC-10AD pump (Shimadzu Scientific Instruments, Columbia, MD).
Compounds were detected and quantified with an ESA coulochem detector
(model 5100A, ESA, Bedford, MA) equipped with conditioning (model 5010, ESA) and microdialysis cells (model 5014B, ESA) (E1 = +0.26 V, E2 = +0.28 V, and guard cell = +0.4 V). The limits of
detection for DA, dihydroxyphenylalanine (DOPA),
dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were 2 pg/20 µl. Peaks were identified by retention times set to known
standards. Data were normalized for total protein for each experiment.
Tissue. Cell monolayers were washed with Dulbecco's
PBS containing Ca2+ and
Mg2+, then covered in 0.1 M perchloric acid. Cells were frozen at  80°C
and then thawed. Samples were collected and sonicated followed by
centrifugation at 15,000 × g for 15 min at 4°C to
remove particulates. Samples were assayed immediately or stored at
80°C until assay.
DA efflux. Cells were rinsed one time with artificial CSF
(ACSF) containing (in mM): 147 NaCl, 2.7 KCl, 1.2 CaCl2, 1.0 MgCl2), then
incubated for 15 min at 37°C to measure basal DA efflux. Samples were
acidified, and particulates were eliminated by centrifugation. Samples
were assayed the same day or stored at 80°C until assay as
described above using 50 µl injections.
DOPA accumulation assay. TH activity was assessed by
measuring the accumulation of DOPA within cells treated with the AADC inhibitor n-hydroxybenzylhydrazine dihydrochloride
(NSD-1015) using a modification of the technique of O'Hara et al.
(1996) and measured by HPLC from 50 µl samples as described above.
Using this method, the rate of intracellular DOPA accumulation is known to be proportional to the rate of tyrosine hydroxylation in
TH-containing cells (J. Waymire, personal observation). Briefly,
triplicate cultures of untransfected MN9D cells, or cells stably
transfected with GFP, wild-type -synuclein, or A53T
-synuclein were grown in 12-well tissue culture plates, washed
two times in ACSF, and equilibrated 20 min at 37°C before addition of
200 µM NSD-1015 in ACSF for 30 min. Parallel
triplicate cultures in ACSF without NSD-1015 served as controls.
Untransfected and GFP-transfected cells were compared with the
wild-type -synuclein or A53T -synuclein biclonal lines in three
to six independent experiments. ACSF samples and tissues were collected
and assayed by HPLC as described above. Data were normalized for total
protein for each condition.
Immunoelectron microscopy
Cells were fixed in cryofix (2% paraformaldehyde, 0.01%
glutaraldehyde in 0.1 M PBS) and stored at 4°C for 1 hr.
Cells were pelleted and resuspended in a small amount of 3% gelatin in
PBS, solidified at 4°C, then fixed an additional 15 min in cryofix. Gelatin-cell block was cryoprotected in polyvinylpyrrolidone (PVP) cryoprotectant overnight at 4°C (25% PVP, 2.3 M sucrose,
0.055 M Na2CO3,
pH 7.4) as described in Tokuyasu (1989). Cell blocks were frozen
on ultracryotome stubs under liquid nitrogen and stored in liquid
nitrogen until use. Ultrathin sections (70-100 nm) were cut using a
Reichert Ultracut U ultramicrotome with a FC4S cryo-attachment, lifted
on a small drop of 2.3 M sucrose, and mounted on
Formvar-coated copper grids. Sections were washed three times with PBS,
then three times with PBS containing 0.5% bovine serum albumin and 0.15% glycine (PBG buffer) followed by a 30 min blocking incubation with 5% normal goat serum in PBG. Sections were labeled with rabbit anti-TH (AB151, Chemicon; 1:500) and mouse anti-synuclein (Syn-1, Transduction Labs; 1:25) in PBG for 1 hr. Sections were washed four
times in PBG and labeled with goat anti-rabbit or goat anti-mouse (5 nm) gold-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ), each at a dilution of 1:25 for 1 hr. Sections were
washed three times in PBG, three times in PBS, then fixed in 2.5%
glutaraldehyde in PBS for 5 min, washed two times in PBS, then washed
six times in ddH2O. Sections were post-stained in 2% neutral uranyl acetate, for 7 min, washed three times in
ddH2O, stained 2 min in 4% uranyl acetate, then
embedded in 1.25% methyl cellulose. Labeling was observed on a JEOL
JEM 1210 electron microscope (Peabody, MA) at 80 kV. This technique,
although superior for immunogold labeling, somewhat compromises
ultrastructural detail (D. Beer-Stolz, personal communication).
In situ DA biosynthesis
We measured the conversion of
L-[1-14C]tyrosine to
14CO2 essentially as
described previously (Waymire et al., 1988). Briefly, cells were grown
on 2- well plates, washed with HEPES-buffered saline (HBS) containing
(in mM): 25 HEPES, 5 glycine, 1 CaCl2, 150 NaCl, 2.0 MgCl2,
0.1 KH2PO4, at pH 7.1, and
incubated with 200 µl HBS containing 50 mCi/mmol radiolabeled
tyrosine for 10-60 min at 37°C. Biosynthesis was stopped by the
addition of 400 µl 10% w/v trichloroacetic acid. A small well
(Kimble/Kontes, Vineland, NJ) was held in place by a rubber stopper
above each culture and contained a folded Whatman paper moistened with
Soluene 350 (Packard Instrument Co., Meriden, CT) for
14CO2 collection.
Radioactivity was measured by liquid scintillation spectroscopy
(Beckman-Coulter LS6500, Fullerton, CA).
Phosphorylation of TH and phosphorimage analysis
Quadruplicate cultures were grown on 24-well Primaria plates
(BD-Falcon Biosciences) in four independent experiments as described above. For phosphorylation, media were replaced with HBS supplemented with 0.5 mCi/ml
32PO4-orthophosphate
to label endogenous ATP by 1.5-4 hr incubation at 37°C.
32P buffer was removed, and cells were
washed and equilibrated for 20 min at 37°C in 200 µl/well HBS.
Duplicate samples of cells were either unstimulated or exposed to 40 mM KCl for 5 min at room temperature to stimulate TH
activity. Cultures were treated with protease inhibitors and frozen at
80°C. Samples were thawed, and lysates were supplemented with
phosphatase inhibitors and processed for TH-IP. For lysates, samples
were separated by SDS-PAGE. Recombinant TH protein (gift of Dr. Paul
Fitzpatrick, Texas A & M University) was used to verify the
identity of the TH bands on Coomassie-stained gels used for
phosphorimage analysis. Proteins from IP samples were separated by
SDS-PAGE and transferred to nitrocellulose membranes. Phosphorylated TH
was quantitated using ImageQuant software (Molecular Dynamics) after
exposure of gels or membranes on a phosphor plate for 3-24 d. Total TH
was determined by immunoblot of the membranes with AB151 anti-TH
antibody (Chemicon).
Protein assay, gel electrophoresis, and immunoblotting
Protein concentrations were determined using BCA relative to BSA
protein standards according to the manufacturer (Pierce, Rockford, IL).
For striatal homogenates and cell lysates, an aliquot of each sample
was reserved as a control. Lysates of MN9D cells were prepared from
cell cultures in 1% NP-40 single detergent buffer containing
leupeptin, aprotinin, and AEBSF at 4°C as described previously (Perez
et al., 1996, 1999). Samples for IP were homogenized; all other lysate
samples were sonicated for ~5 sec using three bursts at an intensity
setting of 4.0, and particulates were eliminated by centrifugation at
17,000 × g for 20 min at 4°C. Supernatants were
assayed immediately or stored at 20°C until assayed. Proteins in
2-4× Laemmli sample buffer were boiled 1 min, separated on Tris-glycine SDS-PAGE (8-15%), and transferred to nitrocellulose membranes. Prestained protein standards (RPN800; Amersham Biosciences) were also used. Equivalent sample loading was verified by 1% Ponceau S
staining before blocking in 5-10% nonfat milk in TBS. Immunoblots were incubated with primary antibodies for 2 hr at room temperature or
overnight at 4°C. After three TBS washes, blots were incubated with
either a peroxidase-coupled anti-mouse or anti-rabbit secondary antibody (Calbiochem, La Jolla, CA) followed by TBS and TBS-Tween (0.05%) washes. Immune complexes were detected by chemiluminescence using SuperSignal (Pierce) or ECL (DuPont NEN, Boston, MA). For some
experiments, linear densitometry of the chemiluminescence signal was
performed using a Kodak Image Station (440 CF; Kodak, Rochester, NY).
TH activity
Tritiated water release from
3,5-3H-L-tyrosine was measured
using modifications of the methods of Nagatsu et al. (1964), Reinhard et al. (1986), Waymire et al. (1991), and Tanji et al. (1994). Using
this assay, 1 mol of
3H2O is generated
for each mole of 3H-L-tyrosine
that is converted to DOPA by TH and is thus a direct measure of TH
activity. Briefly, rat adrenal gland was homogenized in 5 vol of HBS
(see above) containing protease inhibitors and phosphatase inhibitors,
as for co-IP buffer, at 4°C. Particulates were pelleted by
centrifugation at 17,000 × g, and supernatants were
added to an equal volume of 2× assay buffer to bring the final
concentrations to 150 mM Tris maleate, 50 µM L-tyrosine, 5 mM ascorbate, 0.45 mg/ml catalase, and 0.5 mM 6-MPH4 at pH 6.8. Samples were incubated for 5 min at 37°C, and reactions were stopped on ice. Released
3H2O was separated
from unreacted
3H-L-tyrosine by
mixing with 7.5% charcoal/HCl. Released
3H2O was assayed
from the supernatant by liquid scintillation counting. To measure the
impact of -synuclein on TH activity, 0-100
µM purified recombinant human -synuclein
(gift from Ronald Hamilton, University of Pittsburgh) was added to
homogenates before the 5 min incubations at 37°C. BSA at the same
concentrations served as a nonspecific control.
Statistical analyses
ANOVA followed by Tukey-Kramer post hoc comparisons
for ANOVA data significant at 0.05 or better was performed using
Instat3 (Graphpad, San Diego, CA). Data represent the mean ± SEM
from three to eight independent experiments using two to four samples for each condition.
| |
RESULTS |
-Synuclein and TH interact in brain
To evaluate a potential binding interaction between TH and
-synuclein, we prepared rat brain homogenates and performed co-IP assays. Rat striatal samples were immunoprecipitated with antibodies to
-synuclein, TH, or preabsorbed antibodies coupled to protein G beads. An aliquot of homogenate was reserved to reveal the
relative amount of -synuclein (Fig.
1a, lane 1) or TH
(Fig. 1b, lane 1) in the starting samples. Co-IP
with the -synuclein antibody precipitated the expected band at ~19
kDa (Fig. 1a, lane 2) along with an ~60 kDa
protein identified as TH by immunoblotting using the TH antibody (Fig.
1b, lane 2). When the TH antibody was used for
co-IP and proteins were immunoblotted, we observed the expected 60 kDa
TH band (Fig. 1b, lane 3) as well as an ~19 kDa
band identified as -synuclein using an -synuclein-specific
antibody (Fig. 1a, lane 3). Furthermore, the
association between TH and -synuclein was not diminished by
high-stringency co-IP with 500 mM NaCl and 0.1% SDS in control experiments (data not shown). Preincubation of the
-synuclein antibody with recombinant -synuclein protein effectively blocked IP of -synuclein and co-IP of TH (Fig.
1a,b, lane 5). Similarly, incubation
of homogenate with preabsorbed TH antibody (preincubated with
recombinant TH before coupling onto beads) blocked IP of TH and co-IP
of -synuclein (Fig. 1a,b, lane
4). In additional control experiments, we also reconfirmed the association between 14-3-3 and TH (Ostrerova et al., 1999) by co-IP
of striatal homogenates (data not shown). These data identify a
previously undefined association between TH and -synuclein in brain
as confirmed by co-IP using either a TH-specific or an -synuclein-specific antibody and appropriate controls for
specificity.
View larger version (60K):
[in this window]
[in a new window]
|
Figure 1.
Interaction of -synuclein with TH in rat brain.
Data from a representative experiment showing Western blots
(WB) reacted with the -synuclein antibody
(a) or with the TH antibody
(b). a, WB of -synuclein from
rat striatum shows -synuclein in the initial homogenate (lane
1), the -synuclein IP (Syn-IP) sample
(lane 2), the TH-IP sample (lane
3), a control IP using preabsorbed TH antibody (Abs
TH-IP, lane 4), and control IP using
preabsorbed -synuclein antibody (Abs Syn-IP,
lane 5). In b the WB reacted with the TH
antibody shows TH in the initial homogenate (lane 1),
the -synuclein IP sample (lane 2), the TH IP sample
(lane 3), a control IP using preabsorbed TH antibody
(lane 4), and control IP using preabsorbed
-synuclein antibody (lane 5). Nonspecific bands, two
of which appear to be IgG bands, are evident in lanes with stronger
chemiluminescence signal (indicated by asterisks in
a and b). Molecular weights in
kilodaltons (kDa), determined from prestained standards,
are indicated on the left.
|
|
-Synuclein inhibits TH activity in a cell-free assay
Adrenal gland, a rich source of TH, was homogenized and assayed
for TH activity in the presence or absence of recombinant wild-type
-synuclein (0-100 µM) and
3H-L-tyrosine. Whether this
concentration of -synuclein activity is physiologically relevant is
currently unknown. However, the concentrations of -synuclein were
within the range of protein previously used in similar cell-free assays
(Tanji et al., 1994). We observed a dose-dependent decrease in TH
activity as determined by the ability of TH to form
3H2O from
3H-L-tyrosine during a 5 min
incubation, whereas BSA at the same concentrations did not
significantly inhibit TH activity (Fig. 2).
View larger version (43K):
[in this window]
[in a new window]
|
Figure 2.
Inhibition of TH activity by -synuclein
in vitro. Unreacted
3H-L-tyrosine was separated from
3H2O using an acidic charcoal wash, and
3H2O in the resulting supernatants was measured
by scintillation counting for 10 min. Release of
3H2O from 3H-L-tyrosine
was diminished by recombinant -synuclein in a dose-dependent manner
using a cell-free in vitro assay. BSA did not
significantly diminish TH activity at any dose. Data represent the
mean ± SEM for five independent experiments using triplicate
samples for each condition. *p < 0.05;
***p < 0.001.
|
|
MN9D dopaminergic cells as a model for evaluating the interaction
of -synuclein with TH
To generate a model for exploring the physiological relevance of
the impact of -synuclein on TH in neuronal cells, we stably transfected the dopaminergic MN9D line with wild-type or mutant A53T
-synuclein constructs. We also generated stably transfected GFP-expressing cell lines as a control. We observed that GFP-MN9D cells
expressed endogenous levels of -synuclein identical to untransfected
MN9D cells (Fig. 3a,
lanes 1, 2). In contrast, cells stably
transfected with wild-type -synuclein or A53T mutant -synuclein expressed 5-13 times more -synuclein than parental MN9D cells or
control MN9D cells transfected with GFP as determined using linear-range densitometry of immunoblots. Clones expressing similar levels of -synuclein were selected for further characterization (Fig. 3a, lanes 3-6). All MN9D cells
expressed equivalent high endogenous levels of TH as measured by TH
immunoblot analysis (Fig. 3b). Equivalent TH expression
among the cell lines was also confirmed by immunoblots from MN9D cell
extracts normalized for total protein levels (ANOVA; p > 0.12).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 3.
Expression of -synuclein and TH in parental and
in stably transfected MN9D clonal cell lines. a,
Parental MN9D (lane 1) and GFP-transfected MN9D cells
(lane 2) express low endogenous -synuclein levels
compared with clonal cells overexpressing wild-type -synuclein
(WT Syn, lanes 3,
4), or A53T mutant -synuclein (lanes
5, 6) as determined by immunoblot of 20 µg protein per lane reacted with the Syn-1 anti--synuclein
antibody. b, TH levels are equivalent in all MN9D cell
lines whether expressing endogenous -synuclein (parental
MN9D, lane 1, or GFP-MN9D,
lane 2) or overexpressing -synuclein [wild-type
(WT), lanes 3 and
4, or A53T, lanes 5 and
6] as determined by immunoblot of 20 µg
protein per lane reacted with the MAB318 anti-TH antibody.
|
|
Because a loss of viability has been reported in some cells that
overexpress -synuclein (Ostrerova et al., 1999; Zhou et al., 2000),
we also assessed cell viability in our MN9D cell lines. We measured
basal viability using the XTT spectrophotometric assay at equal times
after plating using equal numbers of cells for each condition. We
compared each condition with its own baseline value obtained at 5 hr to
parallel cultures analyzed 24 hr later. We observed that percentage
viability, as determined by XTT, was similar for all MN9D lines.
Parental MN9D cells served as baseline controls (control set to 1.00, with a SEM of ±0.024% between experiments) for comparison with MN9D
GFP (1.05 ± 0.05%), wild-type -synuclein (1.015 ± 0.04%), and A53T -synuclein overexpressing lines (1.16 ± 0.16%; ANOVA, p = 0.72). These data indicate that
-synuclein overexpression in our cells did not significantly
diminish endogenous TH levels or alter basal cell viability.
An association between -synuclein and TH occurs in
MN9D cells
We next determined whether the TH and -synuclein interaction
observed in brain homogenates was also apparent in MN9D cells as
measured by co-IP. Using an -synuclein-specific antibody, we
immunoprecipitated -synuclein from the various MN9D cell lines. As
expected, we were able to immunoprecipitate more -synuclein from
stably transfected -synuclein MN9D lines than from either parental
or GFP transfected control MN9D lines (Fig.
4a). When we probed the co-IP
samples for TH, we observed that TH was immunoprecipitated along with
-synuclein from all cell lines and that more TH was precipitated
with -synuclein in overexpressing cell lines (Fig. 4b),
confirming the association of TH with -synuclein in MN9D cells. In
control experiments we determined that co-IP with a TH-specific
antibody also immunoprecipitated -synuclein from the various cell
lines and that co-IP was eliminated by antibody preabsorption (data not
shown).
View larger version (42K):
[in this window]
[in a new window]
|
Figure 4.
Interaction of -synuclein with TH in MN9D
cells. Western blots (WB) from a representative co-IP
experiment reacted with the anti--synuclein antibody
(a) or with the anti-TH antibody
(b). Immunoprecipitation of -synuclein from
cell extracts (a) resulted in co-IP of TH
(b) from parental MN9D (MN9D),
GFP-transfected MN9D (GFP), wild-type
(WT) -synuclein, and A53T -synuclein
(A53T Syn) cells. Immunoelectron microscopy reveals
colocalization of TH with -synuclein that is apparent on
mitochondria (c) and vesicles (d,
e) in MN9D cells. In c and
d, the larger (10 nm) gold particles label -synuclein
and the smaller (5 nm) particles label TH colocalized on the surface of
a mitochondrion (c) and at the edge of a
vesicular structure (d) in an MN9D cell stably
transfected with A53T -synuclein. In e the large (10 nm) gold particles label TH and the small (5 nm) particles label
-synuclein in the cytoplasm of an MN9D cell stably transfected with
wild-type -synuclein. Arrowheads in
c-e point to colocalized small and large
gold particles. Arrows in d point to the
lipid bilayer of a vesicle decorated with large and small gold
particles. m, Mitochondrion; v, vesicle.
Scale bars, 100 nm.
|
|
To further evaluate the potential association of -synuclein
with TH in MN9D cells, we performed double-label immunoelectron microscopy using antibodies specific for TH and -synuclein.
Parental MN9D, GFP-expressing MN9D, and -synuclein-overexpressing
cells were cryoembedded and sectioned before incubation with both
-synuclein and TH antibodies. We confirmed colocalization of TH with
-synuclein on or near mitochondria (Fig. 4c) and
vesicular membranes (Fig. 4d,e) within the
cytosol of MN9D cells. This distribution is reminiscent of
immunogold-labeled TH observed in rat brain (Glass et al., 2001). In
control MN9D cells, which expressed endogenous levels of -synuclein
and TH, TH was also found colocalized with -synuclein but at lower
overall levels, as expected (data not shown). Specificity of the
labeling reaction was confirmed in control samples from which the
primary antibodies were omitted before incubation with immunogold-labeled secondary antibodies. Taken together, these co-IP
and electron microscopy data reconfirm an association of TH with
-synuclein seen in rat brain (Fig. 1) and strengthen the validity of
MN9D cells as a model system for our studies.
Transfected MN9D cells that overexpress -synuclein have reduced
DA levels
To assess steady-state levels of DA from the various MN9D cells,
we prepared cell extracts and measured DA content by HPLC with
electrochemical detection. Parental MN9D cells and GFP control cells
had similarly high DA levels compared with either wild-type -synuclein or A53T -synuclein clonal lines (Fig.
5a). Steady-state DA levels
were significantly reduced in both wild-type -synuclein and A53T
-synuclein clonal lines (Fig. 5a). DA levels were
fourfold lower in wild-type -synuclein lines (5.3 ± 0.98 ng/mg
protein) and approximately eightfold lower in A53T mutant -synuclein
lines (1.9 ± 0.37 ng/mg protein) compared with parental MN9D
cells (20.8 ± 1.52 ng/mg protein) or GFP cells (22.4 ± 0.75 ng/mg protein; ANOVA; p < 0.0001) (Fig.
5a). DA levels were lower in A53T cells compared with
wild-type cells in this series of experiments; however, post
hoc analyses confirmed that the difference was not statistically significant (p > 0.05). Thus, wild-type and
A53T mutant -synuclein reduced steady-state DA levels similarly,
suggesting that both -synucleins interact with TH to influence DA
synthesis.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 5.
Effect of -synuclein overexpression on cellular
DA and DA efflux from MN9D cells. Supernatants obtained from cell
lysates of parental MN9D, GFP, wild-type -synuclein (WT
SYN), and A53T mutant -synuclein cells were assayed
for DA using HPLC with electrochemical detection
(a). Although A53T cells had even lower cellular
DA levels than wild-type -synuclein cells in a, the
difference was not statistically significant using ANOVA with
Tukey-Kramer post hoc analyses
(p > 0.05). To measure DA efflux from the
various MN9D lines, cells were washed in ACSF and incubated in fresh
ACSF for 15 min at 37°C before collection (b).
Data, normalized for total protein, represent the mean ± SEM of
triplicate samples from two to six independent experiments.
Black bar, MN9D; light gray bar, GFP;
white bar, wild-type -synuclein; dark gray
bar, A53T -synuclein. **p < 0.01;
***p < 0.001.
|
|
The decrease in DA levels observed in MN9D cells overexpressing
-synuclein is consistent with an inhibition of TH; however, there
are other possible explanations. For example, reduced DA levels may
have resulted from enhanced DA degradation. To assess this possibility
we measured DA metabolites DOPAC and HVA by HPLC from MN9D cell
lysates. We observed that although the DOPAC and HVA levels in cell
lysates varied somewhat between experiments, DOPAC and HVA were
consistently higher in lysates from control MN9D cells than in lysates
from stably transfected -synuclein MN9D lines, where they were only
slightly above the limits of detection (data not shown). Thus,
increased degradation of DA did not appear to explain the reduced
intracellular DA in wild-type or A53T mutant -synuclein lines.
-Synuclein binds to lipids and associates with vesicles (Maroteaux
et al., 1988; Davidson et al., 1998; Jensen et al., 1998) (Fig.
4c,d). Thus, another possible explanation for the
reduction in DA levels in cells overexpressing -synuclein is that
the protein increases DA efflux. To evaluate this possibility as a
means of reducing cellular DA, we measured DA efflux from the various
MN9D lines. In three independent experiments using triplicate cultures for each condition, media were replaced with 37°C Dulbecco's
PBS or ACSF, and cells were incubated in these media for 15 min
at 37°C. The new media were collected and acidified, and the amount of DA released from cells during the 15 min incubation period was then
measured (Fig. 5b). The concentration of DA present in media
from wild-type (3.5 ± 0.5 nM) and A53T
mutant (3.2 ± 0.2 nM) -synuclein lines
was ~65% less than in media from untransfected parental MN9D cells
(9.3 ± 1.5 nM) or GFP-transfected MN9D
cells (8.8 ± 0.95 nM) (Fig. 5b).
Similar levels of DA efflux were observed from cells expressing
wild-type or A53T mutant -synuclein, and efflux paralleled
intracellular DA levels in each of the cell lines (ANOVA;
p < 0.001). Taken together, the data suggest that the
differences in DA levels observed from cells overexpressing wild-type
or A53T mutant -synuclein cannot be explained as an increase in DA
catabolism or in DA efflux but instead were the result of reduced DA
biosynthesis perhaps caused by TH inhibition.
DA synthesis is diminished in MN9D cells stably expressing
wild-type or A53T mutant -synuclein
The above data revealed that MN9D cells expressing endogenous
levels of -synuclein had equivalent levels of DA synthesis but that
the rate of DA biosynthesis was reduced in cells overexpressing wild-type or A53T mutant -synuclein. Because parental MN9D and GFP
expressing MN9D lines were essentially identical by all measures, parental MN9D cells were used as controls in subsequent studies. To
directly assess DA biosynthesis as a function of -synuclein expression and TH activity, we used the hydrazine compound NSD-1015 to
inhibit AADC, thereby blocking the conversion of DOPA to DA (see model
in Fig. 8a). Cells were exposed to 200 µM NSD-1015 for 30 min at 37°C, lysates were
collected, and samples were assayed for differences in cellular DOPA
after NSD treatment. We observed that parental MN9D cells significantly
increased DOPA (78.5 ± 21.5%) (Fig.
6a) as compared with untreated
cells. However, in marked contrast, wild-type -synuclein lines
increased their DOPA levels by only 9.0 ± 7.0%, and A53T mutant
-synuclein lines increased DOPA by only 4.0 ± 3.0% (ANOVA;
p < 0.001) compared with untreated cells for each
group. Because DOPA accumulation could only occur if TH actively
converted tyrosine to DOPA, these data suggest that TH activity was
diminished in cells overexpressing either wild-type or mutant
-synuclein.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 6.
Effect of -synuclein overexpression on DA
synthesis in MN9D cell lines. Cells lysates were prepared from
equivalent cultures of MN9D cells incubated in the presence of 200 µm
NSD-1015 for 30 min. a, More DOPA accumulated after
blocking AADC activity with NSD-1015 in parental MN9D cells than in
wild-type (WT) or A53T mutant -synuclein
lines, indicating that TH activity is dramatically reduced in cells
that overexpress -synuclein. b, In
situ DA synthesis was significantly greater for parental MN9D
cells than for WT or A53T -synuclein clonal lines incubated with
L-[1-14C]tyrosine and compared for their
ability to generate 14CO2 from the radioactive
precursor. Data are from two to six independent experiments presented
as the mean ± SEM for each condition. *p < 0.05; **p < 0.01; ***p < 0.001.
|
|
We next assessed DA synthesis in the various MN9D lines by comparing
the conversion of
L-[1-14C]tyrosine with
14CO2, which
requires the activities of both TH and AADC. We observed significantly
less 14CO2 generated
by wild-type or A53T mutant -synuclein MN9D lines when compared with
parental cells (Fig. 6b). In data from eight independent
experiments we observed that wild-type -synuclein expressing cells
produced ~40% less
14CO2 than controls
(p < 0.05), and A53T mutant -synuclein lines produced ~50% less
14CO2 than controls
(p < 0.01). Taken together, the data implicate both wild-type and A53T mutant -synuclein in DA synthesis inhibition.
TH phosphorylation is diminished by overexpression of wild-type or
A53T mutant -synuclein
The above data revealed that DA synthesis was reduced in cells
overexpressing either wild-type or A53T mutant -synuclein. The
short-term regulation of TH activity occurs primarily by changes in TH
phosphorylation. Because 14-3-3 is known to bind to and can sustain the
activity of phosphorylated TH (Itagaki et al., 1999; Muslin and Xing,
2000) and because both 14-3-3 and -synuclein appear to affect the
activities of proteins to which they bind (Ostrerova et al., 1999; Van
Der Hoeven et al., 2000), we next measured TH phosphorylation in our
MN9D lines to determine whether overexpression of -synuclein may
have altered TH phosphorylation. We labeled cells with
32P-orthophosphate and measured
TH-phosphorylation using autoradiography and phosphorimage
analysis. When we compared TH phosphorylation from cell lysates we
observed that cells overexpressing wild-type -synuclein or A53T
mutant -synuclein had 20-30% less phosphorylated TH compared with
controls (ANOVA; p < 0.01) (Fig.
7a). Immunoprecipitation of TH
from the various cell lines brought down similar amounts of TH (Fig.
7b); however, phosphorylated TH levels were significantly reduced in cells that overexpressed -synuclein (Fig. 7c).
Because an equivalent reduction in TH phosphorylation was observed for both wild-type and A53T -synuclein lines, the data suggest that both
forms of -synuclein were able to inhibit TH phosphorylation similarly.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 7.
The impact of -synuclein on TH phosphorylation
in MN9D cells. Cells labeled with 32P were evaluated for TH
phosphorylation by autoradiography of cell lysates from control MN9D
and stably transfected -synuclein lines separated using SDS-PAGE and
analyzed by phosphorimaging (a). After TH
immunoprecipitation from cell lysates, total TH levels (at
arrow) from a representative experiment appear similar
for MN9D, WT, and A53T MN9D lines as determined by Western blot
reacted with the AB151 anti-TH antibody and visualized by
chemiluminescence (b). Phospho-TH levels (at
arrow in c) are significantly reduced in
cells overexpressing wild-type -synuclein or A53T mutant
-synuclein compared with control MN9D cells as seen by
autoradiography. In a, black bar
represents MN9D, white bar represents wild-type
-synuclein, and gray bar represents A53T
-synuclein. Molecular weights, determined from prestained standards,
are indicated on the right in b and
c. *p < 0.05;
**p < 0.01.
|
|
| |
DISCUSSION |
Our studies have allowed us to identify an association between
-synuclein and TH that appears to be functionally significant for DA
neurons. This association, first identified in brain homogenates, prompted us to explore the potential impact of the -synuclein-TH interaction in vitro using a cell-free assay. Addition of
recombinant human -synuclein produced a dose-dependent inhibition of
TH activity. To explore the potential effect of -synuclein on TH in
a more physiological model, we compared control dopaminergic cells
expressing endogenous levels of -synuclein with cell lines stably
transfected to overexpress wild-type or A53T mutant
-synuclein. We confirmed that TH expression was not significantly
altered by -synuclein overexpression in our stably transfected cell
lines and that -synuclein transgene expression was maintained at
high levels during our studies.
The data that we obtained allow us to make three novel observations:
(1) -synuclein interacts with TH in brain as well as in a
dopaminergic cell line, (2) -synuclein inhibits TH activity in a
cell-free assay, and (3) overexpression of -synuclein in a
dopaminergic cell line dramatically reduces TH activity, TH phosphorylation, and DA synthesis.
The region of 14-3-3 that is homologous to -synuclein contains
conserved amino acids that mediate the binding of 14-3-3 to diverse
ligands (Zhang et al., 1997). Thus, assuming that the 14-3-3 homologous
region on -synuclein, which extends from amino acids 1 to 60 in the
amino terminus, subserves protein binding, we anticipated that the A53T
mutation of -synuclein would alter the effect of -synuclein on TH
by altering protein-protein interactions. However, the A53T mutant
-synuclein appeared to behave similarly to wild-type -synuclein
by all measures related to DA synthesis in our studies, strongly
suggesting that both forms of -synuclein interact similarly with TH
to inhibit DA synthesis. Furthermore, although not directly evaluated
in these studies, our findings suggest that the region of -synuclein
involved in its association with TH may not reside in the first 60 amino acids of -synuclein, which harbors both A53T and A30P
mutations. Stefanis and colleagues (2001) recently reported that
overexpressing A53T mutant -synuclein, but not wild-type
-synuclein, in PC12 cells induced a loss of dense-core secretory
granules (DCGs) with a coincident loss of depolarization-evoked DA
release. However, because PC12 cells store DA and other catecholamines
in DCGs, the loss of DCGs would be anticipated to eliminate DA release.
Furthermore, basal DA efflux in the absence of depolarization, as we
measured, was not described for PC12 -synuclein lines, making direct
comparison of our DA data with theirs impossible. In addition,
the A53T PC12 cells exhibited impaired lysosomal/proteosomal function
and reduced cell viability, whereas our -synuclein-expressing MN9D
cell lines were equally viable. Moreover, no loss of nigral DA neurons
is reported for A53T overexpressing transgenic mice (van der Putten et
al., 2000; Matsuoka et al. 2001), suggesting that not all
dopaminergic cells are compromised by A53T -synuclein overexpression.
-Synuclein and 14-3-3 are both enriched in the cytoplasm of nerve
terminals (Maroteaux et al., 1988; Irizarry et al., 1996; Broadie et
al., 1997; Withers et al., 1997; Murphy et al., 2000), the same
compartment that contains TH (Pickel et al., 1975; Glass et al.,
2001) and in which tyrosine hydroxylation takes place. Once
generated, DA is normally packaged into synaptic vesicles by the
activity of the vesicular monoamine transporter, which not only
provides packaged neurotransmitter available for stimulated release
(Nirenberg et al., 1996) but also serves to detoxify the intracellular
environment by removing highly reactive DA from the cytoplasm (Liu et
al., 1992). Thus, if the activities of -synuclein and 14-3-3 are
necessary to maintain normal DA levels, a loss of -synuclein by
decreased expression (Neystat et al., 1999) or aggregation (for review,
see El-Agnaf and Irvine, 2000), should lead to an overproduction of DA
and a subsequent increase in the cytoplasmic concentration of this molecule.
On the basis of previous reports from the literature and our new
findings regarding -synuclein inhibition of TH activity and DA
synthesis, we propose the following model for -synuclein in DA
homeostasis with possible consequences for neurodegeneration (Fig.
8). (1) Under normal circumstances,
14-3-3 binds to phospho-TH to enhance TH activity. Active TH converts
tyrosine to DOPA, which in turn is decarboxylated by AADC to produce
DA. -Synuclein, on the other hand, interacts with TH and inhibits
its activity by decreasing TH phosphorylation. (2) Soluble
-synuclein levels, however, are reduced in PD. The reduction frees
binding partners of -synuclein, such as TH, to bind to 14-3-3 leading to TH activation and increased DA synthesis. (3) If DA levels
exceed the ability of the cells to remove DA from the cytosol, the
accumulated cytosolic DA forms DA-quinone and DA-associated oxyradicals
leading to DA neuronal cell death.
View larger version (30K):
[in this window]
[in a new window]
|
Figure 8.
The potential roles of -synuclein in dopamine
synthesis (a) and neurodegeneration
(b). DA is synthesized in a multistep process in
which the amino acid tyrosine is converted to DOPA by the activity of
phosphorylated TH and aromatic amino acid decarboxylase
(AADC). 14-3-3 is known to bind to TH that is
phosphorylated and -synuclein appears to bind to dephospho-TH.
14-3-3 and -synuclein may differentially regulate TH activity (at
1 in a) to maintain optimal DA levels in
concert with the activities of the vesicular monoamine transporter
(VMAT2, indicated by a small box on
vesicle), which normally packages intracellular DA into vesicles, and
the dopamine transporter (DAT, indicated by large
white box outlined in black on the plasma
membrane), which can function bidirectionally. -Synuclein may
directly bind to TH and inhibit TH activity and/or phosphorylation, or
-synuclein may act indirectly by activating a phosphatase (indicated
by + in a) or inhibiting a kinase (indicted by in a) to affect TH phosphorylation. In b,
a reduction in free soluble -synuclein (indicated by gray
dashed X over -syn, at 2) may
occur by downregulation of -synuclein mRNA or by stimuli that induce
fibrilization (e.g., environmental toxins, mutation, nitration, or
ubiquitination). Disinhibition of TH may lead to elevated cytosolic DA
in neurons with subsequent generation of DA-quinone and DA-related ROS
(at 3 in b), which can damage proteins,
lipids, and DNA and contribute to neurotoxicity.
|
|
Support for our model derives from several observations indicating that
14-3-3 binds to phospho-TH (Ichimura et al., 1988; Kumer and Vrana,
1996; Muslin and Xing, 2000; Kleppe et al., 2001), interaction of TH
with 14-3-3 enhances TH activation (Itagaki et al., 1999) and
phosphorylation (Bevilaqua et al., 2001), cytosolic DA can form
DA-quinone and reactive oxygen species (ROS) that are neurotoxic
(Graham, 1978; Hastings et al., 1996; Stokes et al., 1999; Lotharius
and O'Malley, 2000), -synuclein itself is modified by DA quinone
(Conway et al., 2001), and DA release is enhanced in mice lacking
-synuclein expression as measured by paired-pulse stimulation
(Abeliovich et al., 2000). According to our model, the absence of
-synuclein would increase TH activity and thus provide more DA for
release at the second pulse, which is the case. However, other findings
in the -synuclein knock-out mice appear less supportive of our
model. For example, Abeliovich and colleagues (2000) reported lower
peak levels of extracellular DA from amphetamine-stimulated striatal
slices. Interestingly, substantia nigra DA levels were not reduced in
-synuclein knock-out mice. Perhaps the reduction in striatal DA
occurred secondary to a reduction in the size of the pool of
synaptic vesicles as suggested by the work of Murphy and colleagues
(2000) using an in vitro model. Additionally, DA release was
evoked by amphetamine, which acts to reverse the DA transporter.
Because -synuclein binds to the DA transporter and affects its
activity (Lee et al., 2001), this may also have contributed to
differences in extracellular DA levels from knock-out mice. Finally,
our model proposes that a loss of -synuclein function would lead to
elevated intracellular DA with potential neurotoxic consequences.
-Synuclein knock-out mice have normal numbers of DA neurons at 3-6
weeks (Abeliovich et al., 2000). However, neurotoxicity of DA neurons
secondary to oxyradical damage can be delayed for many months (Ueda et
al., 2000) and thus may not be present after only a few weeks.
Our findings suggest that the impact of -synuclein on TH activity
may be direct, as suggested by the homology of -synuclein with
14-3-3, the ability to co-IP TH with -synuclein from brain and
dopaminergic cells, and the colocalization of both proteins in neuronal
cells. However, it is feasible that the effects of -synuclein are
indirect. One possibility is that -synuclein may inhibit a kinase or
activate a phosphatase involved in TH phosphorylation/dephosphorylation. Alternatively, -synuclein and TH
may be associated within a larger protein complex, and we are further
exploring these possibilities. In any event, our data identify a
functional role for -synuclein in the regulation of DA synthesis
that is maintained by the A53T mutant form of -synuclein. Because
-synuclein is strongly implicated in PD and because DA neurons are
the cells selectively damaged during PD pathogenesis, our findings
provide a focal point for further exploration into the causes and
treatment of the disease. Whether a loss of functional -synuclein is
associated with increased brain DA turnover is not yet known. However,
increased DA turnover in residual DA neurons has long been reported in
patients with even mild forms of PD (Bernheimer et al., 1973).
Elucidation of the potential role of -synuclein in such a process
awaits further analysis.
| |
FOOTNOTES |
Received Dec. 14, 2001; revised Feb. 5, 2002; accepted Feb. 5, 2002.
This work was supported by the Scaife Family Foundation, the National
Parkinson Foundation, and the National Institute of Neurological
Disorders and Stroke (NS19608). We thank Alfred Heller and Lisa Won for
the MN9D cells; Ronald Hamilton and Paul Fitzpatrick for recombinant
proteins; Yong-Jian Liu for -synuclein plasmids; Guodong Cao for the
eGFP plasmid; Simon Watkins and Donna Beer-Stolz for help with electron
microscopy; Alex Glessner and Susan Slagel for expert technical
assistance; and Teresa Hastings for thoughtful critique of this
manuscript. This research is dedicated to D. Beyer, J. Cordy, M. J. Fox, and to the memory of Lester "Rusty" Lanelli.
Correspondence should be addressed to Dr. Ruth G. Perez, Department of
Neurology, Biomedical Science Tower, S510, University of Pittsburgh
School of Medicine, Pittsburgh, PA 15213. E-mail: perezrg{at}pitt.edu.
| |
REFERENCES |
-
Abeliovich A,
Schmitz Y,
Farinas I,
Choi-Lundberg D,
Ho WH,
Castillo PE,
Shinsky N,
Verdugo JM,
Armanini M,
Ryan A,
Hynes M,
Phillips H,
Sulzer D,
Rosenthal A
(2000)
Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system.
Neuron
25:239-252[Web of Science][Medline].
-
Bernheimer H,
Birkmayer W,
Hornykiewicz O,
Jellinger K,
Seitelberger F
(1973)
Brain dopamine and the syndromes of Parkinson and Huntington: clinical, morphological, and neurochemical correlations.
J Neurol Sci
20:415-455[Web of Science][Medline].
-
Betarbet R,
Sherer TB,
MacKenzie G,
Garcia-Osuna M,
Panov AV,
Greenamyre JT
(2000)
Chronic systemic pesticide exposure reproduces features of Parkinson's disease.
Nat Neurosci
3:1301-1306[Web of Science][Medline].
-
Bevilaqua LR,
Graham ME,
Dunkley PR,
von Nagy-Felsobuki EI,
Dickson PW
(2001)
Phosphorylation of ser19 alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of ser40.
J Biol Chem
276:40411-40416[Abstract/Free Full Text].
-
Broadie K,
Rushton E,
Skoulakis EM,
Davis RL
(1997)
Leonardo, a Drosophila 14-3-3 protein involved in learning, regulates presynaptic function.
Neuron
19:391-402[Web of Science][Medline].
-
Brunet A,
Bonni A,
Zigmond MJ,
Lin MZ,
Juo P,
Hu LS,
Anderson MJ,
Arden KC,
Blenis J,
Greenberg ME
(1999)
Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
Cell
96:857-868[Web of Science][Medline].
-
Choi HK,
Won LA,
Kontur PJ,
Hammond DN,
Fox AP,
Wainer BH,
Hoffmann PC,
Heller A
(1991)
Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion.
Brain Res
552:67-76[Web of Science][Medline].
-
Choi HK,
Won L,
Roback JD,
Wainer BH,
Heller A
(1992)
Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions.
Proc Natl Acad Sci USA
89:8943-8947[Abstract/Free Full Text].
-
Conway KA,
Harper JD,
Lansbury PT
(1998)
Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease.
Nat Med
4:1318-1320[Web of Science][Medline].
-
Conway KA,
Lee SJ,
Rochet JC,
Ding TT,
Harper JD,
Williamson RE,
Lansbury Jr PT
(2000)
Accelerated oligomerization by Parkinson's disease linked alpha-synuclein mutants.
Ann NY Acad Sci
920:42-45[Web of Science][Medline].
-
Conway KA,
Rochet JC,
Bieganski RM,
Lansbury Jr PT
(2001)
Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct.
Science
294:1346-1349[Abstract/Free Full Text].
-
Davidson WS,
Jonas A,
Clayton DF,
George JM
(1998)
Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes.
J Biol Chem
273:9443-9449[Abstract/Free Full Text].
-
El-Agnaf OM,
Irvine GB
(2000)
Review: formation and properties of amyloid-like fibrils derived from alpha-synuclein and related proteins.
J Struct Biol
130:300-309[Web of Science][Medline].
-
Feany MB,
Bender WW
(2000)
A Drosophila model of Parkinson's disease.
Nature
404:394-398[Medline].
-
Galvin JE,
Lee VM,
Trojanowski JQ
(2001)
Synucleinopathies: clinical and pathological implications.
Arch Neurol
58:186-190[Abstract/Free Full Text].
-
Giasson BI,
Uryu K,
Trojanowski JQ,
Lee VM
(1999)
Mutant and wildtype human alpha-synucleins assemble into elongated filaments with distinct morphologies in vitro.
J Biol Chem
274:7619-7622[Abstract/Free Full Text].
-
Glass MJ,
Huang J,
Aicher SA,
Milner TA,
Pickel VM
(2001)
Subcellular localization of the -2A-adrenergic receptors in the rat medial nucleus tractus solitarius: regional targeting and relationship with catecholamine neurons.
J Comp Neurol
433:193-207[Web of Science][Medline].
-
Golbe LI,
Di Iorio G,
Sanges G,
Lazzarini AM,
La Sala S,
Bonavita V,
Duvoisin RC
(1996)
Clinical genetic analysis of Parkinson's disease in the Contursi kindred.
Ann Neurol
40:767-775[Web of Science][Medline].
-
Graham DG
(1978)
Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones.
Mol Pharmacol
14:633-643[Abstract/Free Full Text].
-
Hashimoto M,
Hsu LJ,
Sisk A,
Xia Y,
Takeda A,
Sundsmo M,
Masliah E
(1998)
Human recombinant NACP/alpha-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease.
Brain Res
799:301-306[Web of Science][Medline].
-
Hastings TG,
Lewis DA,
Zigmond MJ
(1996)
Role of oxidation in the neurotoxic effects of intrastriatal dopamine injections.
Proc Natl Acad Sci USA
93:1956-1961[Abstract/Free Full Text].
-
Haycock JW
(1990)
Phosphorylation of tyrosine hydroxylase in situ at serine 8, 19, 31, and 40.
J Biol Chem
265:11682-11691[Abstract/Free Full Text].
-
Hornykiewicz O,
Kish SJ
(1986)
Biochemical pathophysiology of Parkinson's disease.
In: Advances in neurology, Vol 45, Parkinson's disease (Yahr MD,
Bergmann KJ,
eds), pp 19-34. New York: Raven.
-
Ichimura T,
Isobe T,
Okuyama T,
Takahashi N,
Araki K,
Kuwano R,
Takahashi Y
(1988)
Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases.
Proc Natl Acad Sci USA
85:7084-7088[Abstract/Free Full Text].
-
Irizarry MC,
Kim TW,
McNamara M,
Tanzi RE,
George JM,
Clayton DF,
Hyman BT
(1996)
Characterization of the precursor protein of the non-A beta component of senile plaques (NACP) in the human central nervous system.
J Neuropathol Exp Neurol
55:889-895[Web of Science][Medline].
-
Itagaki C,
Isobe T,
Taoka M,
Natsume T,
Nomura N,
Horigome T,
Omata S,
Ichinose H,
Nagatsu T,
Greene LA,
Ichimura T
(1999)
Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins.
Biochemistry
38:15673-15680[Medline].
-
Jackson D,
Abercrombie ED,
Zigmond MJ
(1993)
Impact of L-dopa on striatal acetylcholine release: effects of 6-hydroxydopamine.
J Pharmacol Exp Ther
267:912-918[Abstract/Free Full Text].
-
Jenco JM,
Rawlingson A,
Daniels B,
Morris AJ
(1998)
Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by alpha- and beta-synucleins.
Biochemistry
37:4901-4909[Medline].
-
Jenner P,
Olanow CW
(1998)
Understanding cell death in Parkinson's disease.
Ann Neurol
44:S72-84[Web of Science][Medline].
-
Jensen PH,
Nielsen MS,
Jakes R,
Dotti CG,
Goedert M
(1998)
Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation.
J Biol Chem
273:26292-26294[Abstract/Free Full Text].
-
Kim TD,
Paik SR,
Yang CH,
Kim J
(2000)
Structural changes in alpha-synuclein affect its chaperone-like activity in vitro.
Protein Sci
9:2489-2496[Web of Science][Medline].
-
Kleppe R,
Toska K,
Haavik J
(2001)
Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association.
J Neurochem
77:1097-1107[Web of Science][Medline].
-
Kowall NW,
Hantraye P,
Brouillet E,
Beal MF,
McKee AC,
Ferrante RJ
(2000)
MPTP induces alpha-synuclein aggregation in the substantia nigra of baboons.
NeuroReport
11:211-213[Web of Science][Medline].
-
Kruger R,
Kuhn W,
Muller T,
Woitalla D,
Graeber M,
Kosel S,
Przuntek H,
Epplen JT,
Schols L,
Riess O
(1998)
Ala30Pro mutation in the gene encoding -synuclein in Parkinson's disease.
Nat Genet
18:106-108[Web of Science][Medline].
-
Kumer SC,
Vrana KE
(1996)
Intricate regulation of tyrosine hydroxylase activity and gene expression.
J Neurochem
67:443-462[Web of Science][Medline].
-
Lee FJ,
Liu F,
Pristupa ZB,
Niznik HB
(2001)
Direct binding and functional coupling of -synuclein to the dopamine transporters accelerate dopamine-induced apoptosis.
FASEB J
15:916-926[Abstract/Free Full Text].
-
Liu Y,
Peter D,
Roghani A,
Schuldiner S,
Prive GG,
Eisenberg D,
Brecha N,
Edwards RH
(1992)
A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter.
Cell
70:539-551[Web of Science][Medline].
-
Lotharius J,
O'Malley KL
(2000)
The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity.
J Biol Chem
275:38581-38588[Abstract/Free Full Text].
-
Markopoulou K,
Wszolek ZK,
Pfeiffer RF,
Chase BA
(1999)
Reduced expression of the G209A alpha-synuclein allele in familial Parkinsonism.
Ann Neurol
46:374-381[Web of Science][Medline].
-
Maroteaux L,
Campanelli JT,
Scheller RH
(1988)
Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal.
J Neurosci
8:2804-2815[Abstract].
-
Masliah E,
Rockenstein E,
Veinbergs I,
Mallory M,
Hashimoto M,
Takeda A,
Sagara Y,
Sisk A,
Mucke L
(2000)
Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders.
Science
287:1265-1269[Abstract/Free Full Text].
-
Matsuoka Y,
Vila M,
Lincoln S,
McCormack A,
Picciano M,
LaFrancois J,
Yu X,
Dickson D,
Langston WJ,
McGowan E,
Farrer M,
Hardy J,
Duff K,
Przedborski S,
DiMonte DA
(2001)
Lack of nigral pathology in transgenic mice expressing human -synuclein driven by the tyrosine hydroxylase promoter.
Neurobiol Dis
8:535-539[Web of Science][Medline].
-
Murphy DD,
Rueter SM,
Trojanowski JQ,
Lee VM
(2000)
Synucleins are developmentally expressed, and alpha-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons.
J Neurosci
20:3214-3220[Abstract/Free Full Text].
-
Muslin AJ,
Xing H
(2000)
14-3-3 proteins: regulation of subcellular localization by molecular interference.
Cell Signal
12:703-709[Web of Science][Medline].
-
Nagatsu T,
Levitt T,
Udenfriend S
(1964)
Tyrosine hydroxylase: the initial step in norepinephrine biosynthesis.
J Biol Chem
239:2910-2917[Free Full Text].
-
Narhi L,
Wood SJ,
Steavenson S,
Jiang Y,
Wu GM,
Anafi D,
Kaufman SA,
Martin F,
Sitney K,
Denis P,
Louis JC,
Wypych J,
Biere AL,
Citron M
(1999)
Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation.
J Biol Chem
274:9843-9846[Abstract/Free Full Text].
-
Neystat M,
Lynch T,
Przedborski S,
Kholodilov N,
Rzhetskaya M,
Burke R
(1999)
-Synuclein expression in substantia nigra and cortex in Parkinson's disease.
Mov Disord
14:417-422[Web of Science][Medline].
-
Nirenberg MJ,
Chan J,
Liu Y,
Edwards RH,
Pickel VM
(1996)
Ultrastructural localization of the vesicular monoamine transporter-2 in midbrain dopaminergic neurons: potential sites for somatodendritic storage and release of dopamine.
J Neurosci
16:4135-4145[Abstract/Free Full Text].
-
O'Hara CM,
Uhland-Smith A,
O'Malley KL,
Todd RD
(1996)
Inhibition of dopamine synthesis by dopamine D2 and D3 but not D4 receptors.
J Pharmacol Exp Ther
277:186-192[Abstract/Free Full Text].
-
Ostrerova N,
Petrucelli L,
Farrer M,
Mehta N,
Choi P,
Hardy J,
Wolozin B
(1999)
-Synuclein shares physical and functional homology with 14-3-3.
J Neurosci
19:5782-5791[Abstract/Free Full Text].
-
Payton JE,
Perrin RJ,
Wraight L,
Clayton DF,
George JM
(2001)
Anomalous immunoreactivity of the Syn-1 antibody to -synuclein.
Soc Neurosci Abstr
27:93.4.
-
Perez RG,
Squazzo SL,
Koo EH
(1996)
Enhanced release of amyloid -protein from codon 670/671 "Swedish" mutant -amyloid precursor protein occurs in both secretory and endocytic pathways.
J Biol Chem
271:9100-9107[Abstract/Free Full Text].
-
Perez RG,
Soriano S,
Hayes JD,
Ostaszewski B,
Xia W-M,
Selkoe DJ,
Chen X,
Stokin G,
Koo EH
(1999)
Mutagenesis identifies new signals for -amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including A42.
J Biol Chem
274:18851-18856[Abstract/Free Full Text].
-
Pickel VM,
Joh TH,
Reis DJ
(1975)
Ultrastructural localization of tyrosine hydroxylase in noradrenergic neurons of brain.
Proc Natl Acad Sci USA
72:659-663[Abstract/Free Full Text].
-
Polymeropoulos MH,
Lavedan C,
Leroy E,
Ide SE,
Dehejia A,
Dutra A,
Pike B,
Root H,
Rubenstein J,
Boyer R,
Stenroos ES,
Chandrasekharappa S,
Athanassiadou A,
Papapetropoulos T,
Johnson WG,
Lazzarini AM,
Duvoisin RC,
Di Iorio G,
Golbe LI,
Nussbaum RL
(1997)
Mutation in the -synuclein gene identified in families with Parkinson's disease.
Science
276:2045-2047[Abstract/Free Full Text].
-
Reinhard Jr JF,
Smith GK,
Nichol CA
(1986)
A rapid and sensitive assay for tyrosine-3-monooxygenase based upon the release of 3H2O and adsorption of [3H]-tyrosine by charcoal.
Life Sci
39:2185-2189[Web of Science][Medline].
-
Souza JM,
Giasson BI,
Lee VMY,
Ischiropoulos H
(2000)
Chaperone-like activity of synucleins.
FEBS Lett
474:116-119[Web of Science][Medline].
-
Spillantini MG,
Schmidt ML,
Lee VM,
Trojanowski JQ,
Jakes R,
Goedert M
(1997)
-Synuclein in Lewy bodies.
Nature
388:839-840[Medline].
-
Spillantini MG,
Crowther RA,
Jakes R,
Hasegawa M,
Goedert M
(1998)
-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies.
Proc Natl Acad Sci USA
95:6469-6473[Abstract/Free Full Text].
-
Stefanis L,
Larsen KE,
Rideout HJ,
Sulzer D,
Greene LA
(2001)
Expression of A53T mutant but not wild-type -synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death.
J Neurosci
21:9549-9560[Abstract/Free Full Text].
-
Stokes AH,
Hastings TG,
Vrana KE
(1999)
Cytotoxic and genotoxic potential of dopamine.
J Neurosci Res
55:659-665[Web of Science][Medline].
-
Takeda A,
Hashimoto M,
Mallory M,
Sundsumo M,
Hansen L,
Sisk A,
Masliah E
(1998)
Abnormal distribution of the non-Abeta component of Alzheimer's disease amyloid precursor/alpha-synuclein in Lewy body disease as revealed by proteinase K and formic acid pretreatment.
Lab Invest
78:1169-1177[Web of Science][Medline].
-
Tanji M,
Horwitz R,
Rosenfeld G,
Waymire JC
(1994)
Activation of protein kinase C by purified bovine brain 14-3-3: comparison with tyrosine hydroxylase activation.
J Neurochem
63:1908-1916[Web of Science][Medline].
-
Tokuyasu KT
(1989)
Use of poly(vinylpyrrolidone) and poly(vinylalcohol)for cryo-ultramicrotomy.
Histochem J
21:163-171[Web of Science][Medline].
-
Ueda S,
Aikawa M,
Ishizuya-Oka A,
Yamaoka S,
Koibuchi N,
Yoshimoto K
(2000)
Age-related dopamine deficiency in the mesostriatal dopamine system of zitter mutant rats: regional fiber vulnerability in the striatum and the olfactory tubercle.
Neuroscience
95:389-398[Web of Science][Medline].
-
Van Der Hoeven PC,
Van Der Wal JC,
Ruurs P,
Van Blitterswijk WJ
(2000)
Protein kinase C activation by acidic proteins including 14-3-3.
Biochem J
347:781-785.
-
van der Putten H,
Wiederhold KH,
Probst A,
Barbieri S,
Mistl C,
Danner S,
Kauffmann S,
Hofele K,
Spooren WP,
Ruegg MA,
Lin S,
Caroni P,
Sommer B,
Tolnay M,
Bilbe G
(2000)
Neuropathology in mice expressing human alpha-synuclein.
J Neurosci
20:6021-6029[Abstract/Free Full Text].
-
Wagner J,
Palfreyman M,
Zraika M
(1979)
Determination of dopa, dopamine, dopac, epinephrine, norepinephrine, alpha-monofluoromethyldopa and alpha-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection.
J Chromatogr
164:41-54[Web of Science][Medline].
-
Waymire JC,
Johnston JP,
Hummer-Lickteig K,
Lloyd A,
Vigny A,
Craviso GL
(1988)
Phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase. Temporal correlation of acetylcholine's effect on site phosphorylation, enzyme activation, and catecholamine synthesis.
J Biol Chem
263:12439-12447[Abstract/Free Full Text].
-
Waymire JC,
Craviso GL,
Lichteig K,
Johnston JP,
Baldwin C,
Zigmond RE
(1991)
Vasoactive intestinal peptide stimulates catecholamine biosynthesis in isolated adrenal chromaffin cells: evidence for a cyclic AMP-dependent phosphorylation and activation of tyrosine hydroxylase.
J Neurochem
57:1313-1324[Web of Science][Medline].
-
Withers GS,
George JM,
Banker GA,
Clayton DF
(1997)
Delayed localization of synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons.
Brain Res Dev Brain Res
99:87-94[Medline].
-
Zhang L,
Wang H,
Liu D,
Liddington R,
Fu H
(1997)
Raf-1 kinase and exoenzyme S interact with 14-3-3 zeta through a common site involving lysine 49.
J Biol Chem
272:13717-13724[Abstract/Free Full Text].
-
Zhou W,
Hurlbert MS,
Schaack J,
Prasad KN,
Freed CR
(2000)
Overexpression of human alpha-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells.
Brain Res
866:33-43[Web of Science][Medline].
Copyright © 2002 Society for Neuroscience 0270-6474/02/2283090-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
J. Wang, H. Lou, C. J. Pedersen, A. D. Smith, and R. G. Perez
14-3-3{zeta} Contributes to Tyrosine Hydroxylase Activity in MN9D Cells: LOCALIZATION OF DOPAMINE REGULATORY PROTEINS TO MITOCHONDRIA
J. Biol. Chem.,
May 22, 2009;
284(21):
14011 - 14019.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Spanagel
Alcoholism: A Systems Approach From Molecular Physiology to Addictive Behavior
Physiol Rev,
April 1, 2009;
89(2):
649 - 705.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Bisaglia, S. Mammi, and L. Bubacco
Structural insights on physiological functions and pathological effects of {alpha}-synuclein
FASEB J,
February 1, 2009;
23(2):
329 - 340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Orimo, T. Uchihara, A. Nakamura, F. Mori, A. Kakita, K. Wakabayashi, and H. Takahashi
Axonal {alpha}-synuclein aggregates herald centripetal degeneration of cardiac sympathetic nerve in Parkinson's disease
Brain,
March 1, 2008;
131(3):
642 - 650.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Cs. Horvath, G. G. Kovacs, V. Kovari, K. Majtenyi, Y. L. Hurd, and E. Keller
Heroin Abuse Is Characterized by Discrete Mesolimbic Dopamine and Opioid Abnormalities and Exaggerated Nuclear Receptor-Related 1 Transcriptional Decline with Age
J. Neurosci.,
December 5, 2007;
27(49):
13371 - 13375.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Periquet, T. Fulga, L. Myllykangas, M. G. Schlossmacher, and M. B. Feany
Aggregated {alpha}-Synuclein Mediates Dopaminergic Neurotoxicity In Vivo
J. Neurosci.,
March 21, 2007;
27(12):
3338 - 3346.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. E. Larsen, Y. Schmitz, M. D. Troyer, E. Mosharov, P. Dietrich, A. Z. Quazi, M. Savalle, V. Nemani, F. A. Chaudhry, R. H. Edwards, et al.
{alpha}-Synuclein Overexpression in PC12 and Chromaffin Cells Impairs Catecholamine Release by Interfering with a Late Step in Exocytosis.
J. Neurosci.,
November 15, 2006;
26(46):
11915 - 11922.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Kontopoulos, J. D. Parvin, and M. B. Feany
{alpha}-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity
Hum. Mol. Genet.,
October 15, 2006;
15(20):
3012 - 3023.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Urano, N. Hayashi, F. Arisaka, H. Kurita, S. Murata, and H. Ichinose
Molecular mechanism for pterin-mediated inactivation of tyrosine hydroxylase: formation of insoluble aggregates of tyrosine hydroxylase.
J. Biochem.,
April 1, 2006;
139(4):
625 - 635.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-s. Liang, W. Mao, C. Iwai, S. Fukuoka, and S. Y. Stevens
Cardiac sympathetic neuroprotective effect of desipramine in tachycardia-induced cardiomyopathy
Am J Physiol Heart Circ Physiol,
March 1, 2006;
290(3):
H995 - H1003.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kuwahara, A. Koyama, K. Gengyo-Ando, M. Masuda, H. Kowa, M. Tsunoda, S. Mitani, and T. Iwatsubo
Familial Parkinson Mutant {alpha}-Synuclein Causes Dopamine Neuron Dysfunction in Transgenic Caenorhabditis elegans
J. Biol. Chem.,
January 6, 2006;
281(1):
334 - 340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Ved, S. Saha, B. Westlund, C. Perier, L. Burnam, A. Sluder, M. Hoener, C. M. P. Rodrigues, A. Alfonso, C. Steer, et al.
Similar Patterns of Mitochondrial Vulnerability and Rescue Induced by Genetic Modification of {alpha}-Synuclein, Parkin, and DJ-1 in Caenorhabditis elegans
J. Biol. Chem.,
December 30, 2005;
280(52):
42655 - 42668.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Utton, W. J. Noble, J. E. Hill, B. H. Anderton, and D. P. Hanger
Molecular motors implicated in the axonal transport of tau and {alpha}-synuclein
J. Cell Sci.,
October 15, 2005;
118(20):
4645 - 4654.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. M. Peng, R. Tehranian, P. Dietrich, L. Stefanis, and R. G. Perez
{alpha}-Synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells
J. Cell Sci.,
August 1, 2005;
118(15):
3523 - 3530.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Cao, C. C. Gelwix, K. A. Caldwell, and G. A. Caldwell
Torsin-Mediated Protection from Cellular Stress in the Dopaminergic Neurons of Caenorhabditis elegans
J. Neurosci.,
April 13, 2005;
25(15):
3801 - 3812.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bonsch, T. Lederer, U. Reulbach, T. Hothorn, J. Kornhuber, and S. Bleich
Joint analysis of the NACP-REP1 marker within the alpha synuclein gene concludes association with alcohol dependence
Hum. Mol. Genet.,
April 1, 2005;
14(7):
967 - 971.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G.-R. Kweon, J. D. Marks, R. Krencik, E. H. Leung, P. T. Schumacker, K. Hyland, and U. J. Kang
Distinct Mechanisms of Neurodegeneration Induced by Chronic Complex I Inhibition in Dopaminergic and Non-dopaminergic Cells
J. Biol. Chem.,
December 10, 2004;
279(50):
51783 - 51792.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Hodara, E. H. Norris, B. I. Giasson, A. J. Mishizen-Eberz, D. R. Lynch, V. M.-Y. Lee, and H. Ischiropoulos
Functional Consequences of {alpha}-Synuclein Tyrosine Nitration: DIMINISHED BINDING TO LIPID VESICLES AND INCREASED FIBRIL FORMATION
J. Biol. Chem.,
November 12, 2004;
279(46):
47746 - 47753.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Fleming, J. Salcedo, P.-O. Fernagut, E. Rockenstein, E. Masliah, M. S. Levine, and M.-F. Chesselet
Early and Progressive Sensorimotor Anomalies in Mice Overexpressing Wild-Type Human {alpha}-Synuclein
J. Neurosci.,
October 20, 2004;
24(42):
9434 - 9440.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Li, C. Lesuisse, Y. Xu, J. C. Troncoso, D. L. Price, and M. K. Lee
Stabilization of {alpha}-Synuclein Protein with Aging and Familial Parkinson's Disease-Linked A53T Mutation
J. Neurosci.,
August 18, 2004;
24(33):
7400 - 7409.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Nuscher, F. Kamp, T. Mehnert, S. Odoy, C. Haass, P. J. Kahle, and K. Beyer
{alpha}-Synuclein Has a High Affinity for Packing Defects in a Bilayer Membrane: A THERMODYNAMICS STUDY
J. Biol. Chem.,
May 21, 2004;
279(21):
21966 - 21975.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. K. Dougherty and D. K. Morrison
Unlocking the code of 14-3-3
J. Cell Sci.,
April 15, 2004;
117(10):
1875 - 1884.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. RECCHIA, P. DEBETTO, A. NEGRO, D. GUIDOLIN, S. D. SKAPER, and P. GIUSTI
{alpha}-Synuclein and Parkinson's disease
FASEB J,
April 1, 2004;
18(6):
617 - 626.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. SIDHU, C. WERSINGER, and P. VERNIER
Does {alpha}-synuclein modulate dopaminergic synaptic content and tone at the synapse?
FASEB J,
April 1, 2004;
18(6):
637 - 647.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Baucum II, K. S. Rau, E. L. Riddle, G. R. Hanson, and A. E. Fleckenstein
Methamphetamine Increases Dopamine Transporter Higher Molecular Weight Complex Formation via a Dopamine- and Hyperthermia-Associated Mechanism
J. Neurosci.,
March 31, 2004;
24(13):
3436 - 3443.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Zhou and C. R. Freed
Tyrosine-to-Cysteine Modification of Human {alpha}-Synuclein Enhances Protein Aggregation and Cellular Toxicity
J. Biol. Chem.,
March 12, 2004;
279(11):
10128 - 10135.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. R. Saha, J. Hill, M. A. Utton, A. A. Asuni, S. Ackerley, A. J. Grierson, C. C. Miller, A. M. Davies, V. L. Buchman, B. H. Anderton, et al.
Parkinson's disease {alpha}-synuclein mutations exhibit defective axonal transport in cultured neurons
J. Cell Sci.,
March 1, 2004;
117(7):
1017 - 1024.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Baptista, M. R. Cookson, and D. W. Miller
Parkin and {alpha}-Synuclein: Opponent Actions in The Pathogenesis of Parkinson'S Disease
Neuroscientist,
February 1, 2004;
10(1):
63 - 72.
[Abstract]
[PDF]
|
|
|
|
|
|
|
Y. Nagano, H. Yamashita, T. Takahashi, S. Kishida, T. Nakamura, E. Iseki, N. Hattori, Y. Mizuno, A. Kikuchi, and M. Matsumoto
Siah-1 Facilitates Ubiquitination and Degradation of Synphilin-1
J. Biol. Chem.,
December 19, 2003;
278(51):
51504 - 51514.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Liang, J. Spence, L. Liu, W. N. Strother, H. W. Chang, J. A. Ellison, L. Lumeng, T.-K. Li, T. Foroud, and L. G. Carr
alpha -Synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol-preferring and -nonpreferring rats
PNAS,
April 15, 2003;
100(8):
4690 - 4695.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Steece-Collier, E. Maries, and J. H. Kordower
Etiology of Parkinson's disease: Genetics and environment revisited
PNAS,
October 29, 2002;
99(22):
13972 - 13974.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Lotharius and P. Brundin
Impaired dopamine storage resulting from {alpha}-synuclein mutations may contribute to the pathogenesis of Parkinson's disease
Hum. Mol. Genet.,
October 1, 2002;
11(20):
2395 - 2407.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Park, H. Y. Jung, T. D. Kim, J. H. Park, C.-H. Yang, and J. Kim
Distinct Roles of the N-terminal-binding Domain and the C-terminal-solubilizing Domain of alpha -Synuclein, a Molecular Chaperone
J. Biol. Chem.,
August 2, 2002;
277(32):
28512 - 28520.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
