 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 15, 2000, 20(16):6048-6054
The A53T  -Synuclein Mutation Increases Iron-Dependent
Aggregation and Toxicity
Natalie
Ostrerova-Golts1,
Leonard
Petrucelli1,
John
Hardy3,
John M.
Lee1, 2,
Matthew
Farer3, and
Benjamin
Wolozin1
Departments of 1 Pharmacology and
2 Pathology, Loyola University Medical Center, Maywood,
Illinois 60153, and 3 Department of Pharmacology, Mayo
Clinic, Jacksonville, Florida 32224
 |
ABSTRACT |
Parkinson's disease (PD) is the most common motor disorder
affecting the elderly. PD is characterized by the formation of Lewy
bodies and death of dopaminergic neurons. The mechanisms underlying PD
are unknown, but the discoveries that mutations in  -synuclein can
cause familial PD and that -synuclein accumulates in Lewy bodies
suggest that -synuclein participates in the pathophysiology of PD.
Using human BE-M17 neuroblastoma cells overexpressing wild-type, A53T,
or A30P -synuclein, we now show that iron and free radical generators, such as dopamine or hydrogen peroxide, stimulate the production of intracellular aggregates that contain -synuclein and
ubiquitin. The aggregates can be identified by immunocytochemistry, electron microscopy, or the histochemical stain thioflavine S. The amount of aggregation occurring in the cells is dependent on the
amount of -synuclein expressed and the type of -synuclein expressed, with the amount of -synuclein aggregation following a
rank order of A53T > A30P > wild-type > untransfected. In addition to stimulating aggregate formation,
-synuclein also appears to induce toxicity. BE-M17 neuroblastoma
cells overexpressing -synuclein show up to a fourfold increase in
vulnerability to toxicity induced by iron. The vulnerability follows
the same rank order as for aggregation. These data raise the
possibility that -synuclein acts in concert with iron and dopamine
to induce formation of Lewy body pathology in PD and cell death in PD.
Key words:
Parkinson's disease; Lewy body; oxidation; neurodegeneration; ubiquitin; dopamine
| |
INTRODUCTION |
Parkinson's disease (PD) is a
common neurodegenerative disorder. The four primary diagnostic signs of
the illness are resting tremor, bradykinesia, muscular rigidity, and
postural instability. These signs of motor deficiency result from the
loss of dopaminergic neurons in the nigrostriatal system (Gibb and
Lees, 1988 ). The neuropathological hallmark of PD is the Lewy body.
Lewy bodies are intracytoplasmic inclusions that occur in degenerating
neurons, which are composed of a dense core of filamentous and granular material surrounded by radially oriented filaments that have a diameter
of 10-20 nm (Goedert et al., 1999). In general, the causes of PD are
not known, and there has been vigorous debate over the relative roles
of genetics and environmental factors (Tanner et al., 1999). The only
defined causes of the disease are A53T and A30P mutations in the
-synuclein gene (Polymeropoulos et al., 1997; Goedert et al., 1999;
Papadimitriou et al., 1999), but there has been much circumstantial
evidence implicating oxidative stress in the etiology of the disease
(Jenner and Olanow, 1998). Immunohistochemical studies indicate that
Lewy bodies stain strongly for -synuclein and ubiquitin (Spillantini
et al., 1997; Jenner and Olanow, 1998; Spillantini et al., 1998;
Markopoulou et al., 1999). The mechanisms by which mutations in
-synuclein lead to PD are unknown. One study suggest that the
mutations might reduce -synuclein expression (Markopoulou et al.,
1999). However, most in vitro experiments using recombinant
protein suggest that the mutations lead to increases in -synuclein
aggregation because of an increase in the rate of aggregation of
mutant A53T and A30P proteins compared with wild-type -synuclein
(Conway et al., 1998; Hashimoto et al., 1998; Giasson et al., 1999).
These findings suggest that increased rate of -synuclein aggregation
might contribute to the mechanisms of neurodegeneration in PD.
Recently, oxidative stress produced by iron and hydrogen peroxide has
been shown to induce amyloid-like aggregate formation of -synuclein
in vitro (Hashimoto et al., 1999; Paik et al., 1999).
Oxidative stress is thought to contribute to PD because dopamine, which
is a strong free radical generator, is the principle neurotransmitter
in the substantia nigra (Chiueh et al., 1993; Jenner and Olanow, 1998).
In addition, iron, which also stimulates free radical production,
accumulates in the substantia nigra with age (Jenner and Olanow, 1998).
Thus, the oxidative conditions present in the substantia nigra could
promote -synuclein aggregation. Whether such oxidative conditions
actually promote -synuclein aggregation in living neurons, however,
is unknown.
| |
MATERIALS AND METHODS |
Materials. -Synuclein (wild-type, A53T, and A30P)
was cloned into the NotI site of pcDNA3. The sequence of
each construct was confirmed by DNA sequencing. For production of
recombinant protein, -synuclein was inserted into the
NcoI/NotI site of the Pro-Ex His-6 vector (Life
Technologies, Gaithersburg, MD). To generate recombinant
-synuclein, BPer (Pierce, Rockford, IL) reagent was used to
solubilize the recombinant -synuclein from the
isopropylthio- -D-galactoside-induced bacterial
lysates, which were then passed over a nickel-agarose affinity column,
washed, and eluted with imidazole according to the directions of the
manufacturer (Life Technologies). After purification, the His-6
tag was cleaved with tobacco etch virus protease and removed by
passing through a nickel-agarose column. Antibodies used include the
following: polyclonal anti--synuclein (SC1; 1:2000 for
immunoblotting and 1:500 for immunocytochemistry against human
-synuclein; residues 116-131; sequence, MPVDPDNEAYEMPSEE),
monoclonal anti--synuclein-1 (1:1000; Transduction Laboratories,
Lexington, KY), and polyclonal rabbit anti-ubiquitin (1:1000 for
immunoblotting and 1:500 for immunocytochemistry; Dako, High Wycombe, UK).
Cell culture. Cells were grown in OPTIMEM (Life
Technologies) supplemented with 10% FBS, nonessential amino acids,
sodium pyruvate, and 500 µg/ml G418, as needed. G418 was used for selection.
Immunoblotting. Cells were harvested with SDS lysis solution
[2% SDS, 10 mM Tris, pH 7.4, 2 mM -glycerol phosphate, and 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride]. The amount of protein was determined using the
BCA assay (Pierce); 5-30 µg/lane was run on 14% SDS polyacrylamide
gels and transferred to nitrocellulose (200 mA, 12 hr). The
nitrocellulose was then incubated 1 hr in 5% milk-PBS, washed,
incubated overnight in 1° antibody, washed, then incubated 3 hr in
peroxidase-coupled 2° antibody, and developed with chemiluminescent
reagent (NEN, Boston, MA).
Cell fractionation. For cell fractionation, the cells were
harvested in buffer containing 20 mM Tris, pH
7.4, 2 mM EDTA, 0.25 M
sucrose, and 20 µg/ml protease inhibitor cocktail (Sigma, St. Louis,
MO). The cell lysate was sonicated and centrifuged at 100,000 × g at 4°C for 1 hr.
MTT and lactate dehydrogenase toxicity assay. Cells were
plated in 96 well dishes at 5000 cells per well in 100 µl of growth medium. For the MTT assay, viability after 48 hr of pharmacological treatment was analyzed by adding 0.5 mg/ml MTT and incubating at
37°C for 3 hr. Lysis buffer (100 µl of 20% SDS in 50%
N,N-dimethylformamide) was then
added, and the plates are read at 540 nm after 24 hr. For the lactate
dehydrogenase (LDH) assay, viability after 24 hr of pharmacological
treatment was analyzed using MTS reagent and the Cytox 96 kit (Promega,
Madison, WI) according to the directions of the manufacturer.
Thioflavine S histochemistry. Cells were fixed 30 min in 4%
paraformaldehyde. After two PBS washes, the cells were incubated with
0.5% thioflavine S for 8 min, washed three times in 80% ethanol, washed once in H2O, and then mounted.
Electron microscopy. Cells were detached by scraping, spun
down, fixed in 2% glutaraldehyde for 2 hr at 4°C, and then
post-fixed in 1% osmium tetroxide for 1 hr at 4°C. The samples were
dehydrated, embedded in epoxy resin (Electron Microscopy Sciences, Fort
Washington, PA), and cut into 70 nm sections for microscopy. The
sections were then post-stained with 5% uranyl acetate and Reynolds
lead citrate. Samples were viewed with a Hitachi H-600 transmission electron microscope at 75 kV.
Iron staining. Cells were fixed 30 min in 4%
paraformaldehyde, followed by two PBS washes, and stained using the
Accustain Iron Stain according to the directions of the manufacturer (Sigma).
Immunohistochemistry. For light microscopy, cells were fixed
with 4% paraformaldehyde, washed, permeabilized by incubation for 30 min with 0.2% Triton X-100, blocked with 5% dry milk-1% goat
serum-PBS, washed, and then incubated overnight in 1° antibody (1:500). Development was with an ABC kit and 3',3'-diaminobenzidine as
per the directions of the manufacturer (Vector Laboratories, Burlingame, CA).
| |
RESULTS |
Iron and free radicals stimulate -synuclein aggregation
To test whether A53T and A30P mutations in -synuclein increase
the tendency of -synuclein to aggregate in neurons, we examined -synuclein aggregation in human BE-M17 neuroblastoma cells stably transfected with wild-type, A53T, or A30P -synuclein (Fig.
1A). Each cell line was
treated for 48 hr with freshly prepared FeCl2 (1 or 10 mM) and then harvested, homogenized, and
fractionated into membrane and cytoplasmic components. The membrane (5 µg/lane) and cytoplasmic (20 µg/lane) components were immunoblotted
with monoclonal anti--synuclein antibody. Aggregates of
-synuclein were evident in the membrane fraction but not in the
cytoplasmic fraction (Fig. 1, membrane fraction shown). Treatment of
the A53T-expressing cell line with FeCl2 induced
dose-dependent formation of heterogeneous high molecular weight
-synuclein aggregates that migrated in the stacking gel (Fig.
1B, solid bracket). A large amount of
anti--synuclein immunoreactivity was also apparent in the upper
portions of the separating gel, in the range of 45-200 kDa (Fig.
1B, lane 2, dotted bracket).
Because these bands are significantly larger than monomeric -synuclein, which has a mass of 19 kDa, these bands might also represent -synuclein polymers and aggregates. For instance, the bands at 38 and 57 kDa have sizes consistent with dimers and trimers of
-synuclein. Immunoblots done with a different antibody, a polyclonal
anti--synuclein antibody, also showed aggregate production under the
same conditions (data not shown). Treatment of the other cell lines
(untransfected, wild-type, and A30P) did not induce -synuclein
aggregates within the 48 hr time frame examined (data not shown).
View larger version (76K):
[in this window]
[in a new window]
|
Figure 1.
Iron stimulates -synuclein aggregation.
A, Immunoblot of -synuclein in cell lines
overexpressing wild-type (WT), A53T, and A30P
-synuclein using the monoclonal anti--synuclein antibody
(top). The same immunoblot was then stripped and
reprobed with antibody to actin. UT, Untransfected.
B, Aggregation of -synuclein in BE-M17 cells
expressing A53T -synuclein after treatment with 1 or 10 mM FeCl2 for 48 hr (in each panel, the
solid bracket shows putative aggregates in the stacking
gel, and the dotted bracket shows putative aggregates in
the separating gel). C, Longer exposure (4 d) enabled
lower doses of FeCl2 to induce aggregation of -synuclein
BE-M17 cells expressing A53T -synuclein (left) or
wild-type -synuclein (middle). Under these
conditions, little or no aggregation of actin was observed
(right). D, In vitro
analysis of -synuclein aggregation. Immunoblotting of -synuclein
in brain membrane fractions under basal conditions (lane
1) or after treatment with 10 mM FeCl2
and 500 µM dopamine (DA) for 24 hr
(lane 2) shows the induction of high molecular weight
aggregates similar to that seen in the BE-M17 cells.
|
|
To determine whether increasing the duration of exposure to iron
produced aggregation of -synuclein at lower doses of iron, we
exposed BE-M17 cells overexpressing A53T or wild-type -synuclein to
FeCl2 for 4 d. Under these conditions, doses
as low as 0.3 µM FeCl2 produced
detectable aggregation of -synuclein in cells expressing A53T
-synuclein (Fig. 1C, left panel).
Interestingly, these longer conditions also induced aggregation of
wild-type -synuclein (Fig. 1C, middle
panel). In contrast, no aggregation of actin was observed,
which suggests that aggregation is selective for -synuclein (Fig.
1C, right panel). We also examined whether Bcl-2-associated death protein (BAD) aggregated, because BAD is a pro-apoptotic protein that binds to -synuclein (Osterova et al.,
1999). However, no aggregated BAD reactivity was apparent (data
not shown).
To understand how the pattern of -synuclein aggregation in the
BE-M17 cells compares with that occurring in human brain, we
investigated the response of -synuclein in human cortical brain
homogenates (from a neurologically normal donor) to iron exposure
in vitro. The pattern of -synuclein aggregation in the membrane fraction of the cortical brain homogenate after treatment for
24 hr with 500 µM dopamine, 10 mM FeCl2, and protease
inhibitors (dopamine was added as an oxidant, as described below) was
similar to that seen in the BE-M17 cells. This suggests that
-synuclein in BE-M17 cells and in human brain exhibit similar
aggregation patterns in response to iron, and both share a strong
tendency to aggregate.
In the course of our investigations, we noted that higher molecular
weight -synuclein immunoreactivity occasionally appeared in
separating gels of immunoblots of -synuclein cell lysates from cells
grown under basal conditions (Fig. 1C). However, aggregates that migrated in the stacking gel only occurred after treatment with
iron and were never observed in any of the cell lines under basal
conditions. This supports previous work done with recombinant -synuclein in vitro indicating that the mutant forms of
-synuclein have a strong tendency to oligomerize (Conway et
al., 2000). However, migration of aggregates in the stacking gel might
be a stricter test of aggregate formation than migration in the
separating gel.
Iron might promote protein aggregation by increasing free radical
formation through the Fenton reaction (Wolozin and Behl, 2000). If so,
then adding free radical generators, such as hydrogen peroxide or
dopamine, along with the iron might increase the amount of
-synuclein aggregation. To test whether oxidation enhanced iron-induced aggregation of -synuclein, we treated BE-M17 cells overexpressing A30P or wild-type -synuclein for 48 hr with 10 mM FeCl2 plus varying concentrations
of dopamine (Fig. 2A,
A30P overexpressing cells shown). We used cells expressing A30P or wild-type -synuclein over 48 hr because they do not form aggregates under these conditions, unlike cells overexpressing A53T -synuclein. As expected, BE-M17 cells overexpressing A30P or wild-type
-synuclein treated with 10 mM
FeCl2 alone did not induce any aggregation (Fig.
2A, lane 2). Similarly, treatment with 5, 50, or 500 µM dopamine alone did not induce
formation of large -synuclein aggregates that migrate in the
stacking gel (Fig. 2A, lanes 3,
5, 7). However, combining 10 mM FeCl2 with 50 or 500 µM dopamine induced formation of large
-synuclein aggregates (Fig. 2A, lanes
6, 8). These data show that the combination of oxidants
and iron can exert additive effects on -synuclein aggregation.
View larger version (30K):
[in this window]
[in a new window]
|
Figure 2.
Free radicals potentiate induction of
-synuclein aggregation by iron. A, B,
Aggregation of -synuclein in BE-M17 cells expressing A30P
-synuclein after treatment with 0, 5, 50, and 500 µM
dopamine plus or minus 10 mM FeCl2 for 48 hr.
The immunoblot was first probed with monoclonal anti-synuclein antibody
(A) and then reprobed with anti-ubiquitin
antibody (B). C, Immunoblots of
lysates from primary cortical neurons after treatment with 0, 0.1, or
10 mM FeCl2 and 50 µM dopamine
(DA) for 60 hr.
|
|
Two separate experiments indicated that the aggregation observed was a
general property of -synuclein rather than an artifact resulting
from use of clonal cell lines. BE-M17 cells that were transiently
transfected with A53T -synuclein cDNA and then treated with 10 mM FeCl2 plus 100 µM
H2O2 for 72 hr developed
aggregates similar to those seen in the BE-M17 cell lines stably
overexpressing -synuclein (data not shown). In addition, primary rat
cortical neurons also showed a strong tendency to develop aggregates,
requiring only 60 hr of treatment with 0.1 mM
FeCl2 and 50 µM dopamine to induce
formation of aggregates (Fig. 2C). Thus, the aggregation was
a result of a biophysical property of -synuclein rather than being
an artifact specific to particular clonal cell lines.
A53T and A30P -synuclein aggregates contain ubiquitin
In PD, Lewy bodies have also been shown to contain large amounts
of ubiquitin (Gibb and Lees, 1988; Dickson et al., 1999). The
presence of ubiquitin in Lewy bodies prompted us to examine whether
aggregation of ubiquitin also occurred along with -synuclein aggregation. We took lysates (membrane fractions) from the BE-M17 cells
expressing A30P -synuclein that had been treated with
FeCl2 and dopamine (Fig. 2A)
and immunoblotted them with anti-ubiquitin antibody (Fig.
2B). The ubiquitin aggregates that accumulated under
these conditions stained strongly for ubiquitin (Fig.
2A,B). Aggregates that accumulated
in cells expressing A53T -synuclein or wild-type -synuclein after
being treated with FeCl2 alone, FeCl2 plus hydrogen peroxide, or
FeCl2 plus dopamine also contained ubiquitin
(data not shown). In many cases, ubiquitin appeared to be a more
sensitive indicator of aggregation on the immunoblots than
-synuclein (Fig. 2B). The amount of
aggregated ubiquitin generally paralleled the amount of -synuclein
(Fig. 2A,B). The presence of
ubiquitin in aggregates did not appear to result from increased
ubiquitin expression because immunoblots of total lysates showed that
the total amount of ubiquitin and actin did not different significantly
between cell lines overexpressing -synuclein and control cells.
These data suggest that aggregates of -synuclein that form in
neurons in response to iron treatment are ubiquinated.
-Synuclein aggregates form visible inclusions evident by
thioflavine S histochemistry and electron microscopy
Next, we used thioflavine S histochemistry and electron microscopy
to examine the aggregates that formed in response to treatment with
iron and hydrogen peroxide. We treated cells from each line (BE-M17:
untransfected, wild-type, A30P, and A53T -synuclein) with 10 mM FeCl2 or 10 mM
FeCl2 plus 100 µM
H2O2 for 72 hr to induce
formation of -synuclein-positive inclusions. The aggregates that
formed were observed to bind thioflavine S, which indicates the
presence of -pleated sheet structures (Fig.
3). The size and number of thioflavine
S-positive aggregates paralleled the results seen by immunoblotting.
The wild-type, A30P, and A53T -synuclein cell lines each showed
significant accumulations of protein aggregates after treatment for 72 hr with 10 mM FeCl2 plus 100 µM H2O2 (Fig.
3A,B). In contrast, the
untransfected line showed no accumulation of thioflavine S-positive
aggregates (Fig. 3C). When only 10 mM
FeCl2 was used to treat the cells, shown thioflavine S-positive inclusions were observed only in A53T
-synuclein-expressing cells (Fig. 3D-F).
View larger version (34K):
[in this window]
[in a new window]
|
Figure 3.
Identifcation of -synuclein aggregates by
thioflavine S staining and by electron microscopy. A-C,
Treatment of cells expressing A53T -synuclein
(A), wild-type -synuclein
(B), or untransfected cells
(C) with 10 mM FeCl2 and
100 µM H2O2 for 72 hr.
D-F, Treatment of cells expressing A53T -synuclein
(D), wild-type -synuclein
(E), or untransfected cells
(F) with 10 mM FeCl2 for
72 hr. G, H, Inclusions are evident by
electron microscopy. BE-M17 cells stably transfected with A53T
-synuclein were treated with 10 mM FeCl2 and
100 µM H2O2 for 72 hr and then
examined by electron microscopy. The aggregates that formed under these
conditions were long fibrils with a diameter of ~10 nm
(G, magnification of 35,000×). Although treatment with
10 mM FeCl2 and 100 µM
H2O2 was toxic to many cells, some cells
containing aggregates had both fibrillar deposits
(triangle) and organelles that were intact
(arrow), suggesting that aggregation can occur in living
cells (H, magnification of 20,000×).
Arrows point to thioflavine S-positive aggregates.
|
|
Next, we used electron microscopy to examine the aggregates. Cells
expressing A53T -synuclein or empty vector were treated with 10 mM FeCl2 plus 100 µM
H2O2 for 72 hr and then
prepared for electron microscopy. Cytoplasmic inclusions were apparent in the A53T-expressing cells but not in the vector-transfected cells
(Fig. 3G,H). The inclusions contained
mixtures of fibrillar and amorphous material (Fig. 3G). The
fibrils had an approximate diameter of 10 nm and a length of up to 10 µm. The presence of both fibrillar and amorphous material in the
aggregates has been observed in aggregates present in transgenic
animals overexpressing -synuclein (Feany and Bender, 2000; Masliah
et al., 2000).
-Synuclein aggregates form visible inclusions evident
by immunohistochemistry
We also examined the inclusion formation by immunocytochemistry.
Cells expressing A53T -synuclein were treated with 10 mM FeCl2 plus 100 µM
H2O2 for 72 hr, fixed, and
then examined with antibodies to ubiquitin and -synuclein using
peroxidase immunohistochemistry. We used peroxidase staining because
fluorescent chromagens, such as FITC or rhodamine, exhibited strong
nonspecific binding to the cells because of the treatment with iron.
Immunohistochemistry with both the anti--synuclein and
anti-ubiquitin antibodies showed uniform staining throughout the
cytoplasm of the cells under basal conditions (Fig.
4A,B).
After treatment with 10 mM
FeCl2 plus 100 µM
H2O2 for 72 hr, the
staining became less uniform. Cells stained with anti--synuclein
antibody often displayed several large darkly stained reactive foci and
multiple small punctate foci in each cell (Fig. 4C).
Staining with ubiquitin was also present but showed foci per cell (Fig.
4D). Immunocytochemistry performed with the preimmune
rabbit serum instead of primary antibody showed no reactivity under
either basal or treated conditions (Fig.
4E,F).
View larger version (35K):
[in this window]
[in a new window]
|
Figure 4.
Identification of -synuclein aggregates by
immunocytochemistry. A, B, Under basal
conditions, BE-M17 cells overexpressing A53T -synuclein showed
diffuse cytoplasmic staining with antibodies to -synuclein
(A) and ubiquitin (B).
C, D, Treatment with 10 mM
FeCl2 and 100 µM H2O2
for 72 hr induced formation of aggregates, which stained positive using
antibodies to -synuclein (C) and ubiquitin
(D). Arrows point to some of the
labeled inclusions. E, In contrast, cells treated with
10 mM FeCl2 and 100 µM
H2O2 for 72 hr and stained with preimmune serum
showed very little reactivity. F, Untreated cells
also showed no reactivity with preimmune serum.
|
|
-Synuclein aggregation occurs in viable cells
Treating cells with FeCl2 can be toxic, and
it is possible that aggregation of -synuclein occurs after cell
death. To determine the relationship between -synuclein aggregation
and cell death, we examined aggregate formation and cell viability in
the presence of high and low concentrations of
FeCl2. As described above (Figs. 3, 4), treatment
with 10 mM FeCl2 plus 100 µM H2O2 for
72 hr induced formation of -synuclein aggregates in most cells;
however, these conditions also were observed to kill most of the cells
(>90% cell death).
In contrast, A53T-expressing BE-M17 cells treated with 0.3 mM FeCl2 plus 100 µM
H2O2 for 96 hr exhibited
much less toxicity, yet still formed -synuclein aggregates. Parallel
sets of cells were processed for immunocytochemistry with antibodies to
-synuclein (SC1) and ubiquitin or were processed to measure
viability using the trypan blue exclusion assay. Untreated cells showed
little toxicity, with 3.7 ± 1.0% being permeable to trypan blue
(Fig. 5). The small amount of cell death
present might have been caused by the trypsinization-trituration step
used to dislodge the cells. Treatment with 0.3 mM
FeCl2 with or without 100 µM
H2O2 killed some cells
(12.1 ± 2.3% trypan blue-positive), but the large majority of
cells (87.9%) remained viable (Fig. 5A).
Immunocytochemistry with anti--synuclein antibody showed that
21.0 ± 3.5% of the cells had visible aggregates (Fig.
5B). Immunocytochemistry with anti-ubiquitin antibody
suggested that the aggregates could contain ubiquitin (Fig.
5C) and that the aggregates contained material that had a
-pleated sheet structure that stained with thioflavine S (Fig.
5D). Interestingly, diffuse thioflavine S reactivity was also evident in these cells, suggesting that dispersed
"micro-aggregates" of -synuclein might also form under the mild
conditions.
View larger version (49K):
[in this window]
[in a new window]
|
Figure 5.
-Synuclein aggregation can occur in viable
cells. BE-M17 cells overexpressing A53T -synuclein were treated with
0.3 mM FeCl2 with or without 100 µM H2O2 for 96 hr.
A, Comparison of cell death and aggregate formation in
cells expressing A53T -synuclein. Cell viability measurements were
based on a trypan blue exclusion assay, and -synuclein aggregates
were identified by immunocytochemistry using the anti--synuclein SC1
antibody. The cell death determinations were based on quantitation of
three different samples of treated and untreated cells. Quantitation of
aggregates was based on counting the number of cells containing
-synuclein aggregates in five microscopy fields chosen at random for
treated and untreated cells. *p < 0.05, **p < 0.001. B, C,
Immunocytochemistry of A53T -synuclein cells treated with 0.3 mM FeCl2 with or without 100 µM
H2O2 for 96 hr. B shows
aggregates that were positive for -synuclein (SC1 antibody), and
C shows aggregates that were positive for ubiquitin
(anti-ubiquitin antibody). D, A53T -synuclein cells
under basal conditions show little staining with thioflavine S. E, Treating A53T -synuclein cells with 0.3 mM FeCl2 for 4 d induced formation of
inclusions that could be stained with thioflavine S. Arrows point to some of the thioflavine S-positive
inclusions. In addition, treated cells (E)
stained much stronger with thioflavine S than untreated cells
(D).
|
|
Thus, the -synuclein aggregates that formed under mild conditions
reacted with the same antibodies and stains as aggregates that formed
under harsher conditions. Moreover, the observation that the percentage
of cells displaying -synuclein aggregates was greater than the
percentage of cells showing evidence of death suggests that many of the
cells developing -synuclein aggregates are viable. The observation
that aggregation of -synuclein can occur in viable cells also
suggests that aggregation of -synuclein precedes cell death.
Overexpression of -synuclein increases free
radical-mediated toxicity
Despite the fact that -synuclein aggregates can form in viable
cells, it is possible that aggregation of -synuclein might represent
an initial step in the induction of toxicity. Our previous results show
that -synuclein is toxic to some cells when transiently overexpressed (Ostrerova et al., 1999). However, -synuclein is not
acutely toxic to all cells. -Synuclein is not acutely toxic to
BE-M17 cells (data not shown), and -synuclein is tolerated well
enough to allow overexpression in transgenic animals (Feany and Bender,
2000; Masliah et al., 2000). Although -synuclein is not acutely
toxic to BE-M17 cells under basal conditions, we hypothesized that
-synuclein might be toxic under other conditions, such as conditions
linked to formation of aggregates.
To determine whether conditions that produce -synuclein aggregation
also produce toxicity, we examined the vulnerability of each cell line
to iron- and/or hydrogen peroxide-mediated toxicity. Each cell line
(untransfected, wild-type, A53T, and A30P -synuclein) was treated
with varying doses of FeCl2,
H2O2, or
FeCl2 plus
H2O2, and the amount of
toxicity was determined by MTT assay. BE-M17 cells overexpressing all
forms of -synuclein showed increased vulnerability to iron-mediated
toxicity (Fig. 6A).
Overexpression of A53T -synuclein had the greatest effect on
toxicity, reducing the LD50 of
FeCl2 over 75% (Fig. 6A).
Overexpression of A30P or wild-type -synuclein constructs reduced
the LD50 values by ~50%, although the amount
of toxicity seen in A30P-expressing cells in response to low levels of
iron was generally greater than toxicity seen with wild-type
-synuclein (Fig. 6A). To confirm that
overexpression of -synuclein increased the vulnerability of the
neuroblastoma cells to iron, we also examined iron-induced toxicity
using an LDH assay (Fig. 6B). The LDH assay
confirmed that overexpression of the -synuclein constructs increases
iron-induced toxicity (Fig. 6B). MTT assays of cell
lines overexpressing -synuclein (wild-type, A53T, or A30P) also
revealed increased toxicity after treatment with
FeCl2 plus
H2O2 (Fig. 6C,
dark gray bars). Interestingly, overexpressing -synuclein
(wild-type, A53T, or A30P) did not increase the vulnerability to
H2O2 alone and conferred
modest protection to the neuroblastoma cells (Fig. 6C).
These data demonstrate that increased levels of either wild-type or
mutant -synuclein can be toxic to neurons grown in cell culture
under selective conditions. The data suggest that -synuclein renders
the cells particularly vulnerable to iron-mediated toxicity. The
selective vulnerability to iron could result from a tendency of
-synuclein to sequester iron in -synuclein aggregates. To test
this, we treated untransfected or A53T -synuclein-expressing BE-M17
cells with 10 mM FeCl2 and
100 µM
H2O2 for 48 hr, fixed them,
and then stained the cells for iron. The cells expressing A53T
-synuclein had a much higher iron content than the untransfected
cells after treatment (Fig. 6D). Sequestration of
iron attributable to -synuclein would increase free radical
production via the Fenton reaction, particularly in cells exposed to
free radical generators, such as hydrogen peroxide or dopamine.
However, in the absence of iron, no Fenton reaction occurs and the
-synuclein is innocuous.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 6.
-Synuclein increases iron-dependent toxicity.
A, B, BE-M17 cells overexpressing
wild-type, A53T, or A30P -synuclein were treated with varying doses
of FeCl2 for 48 hr, and the viability was determined using
the MTT assay (A) or the LDH assay
(B). C, BE-M17 cells
overexpressing wild-type, A53T, or A30P -synuclein were treated with
varying doses of H2O2 for 48 hr, and the
viability was determined using the MTT assay. *p < 0.001, +p < 0.01 by ANOVA analysis.
D, Iron stain of BE-M17 cells. a,
Untransfected cells, basal conditions; b, A53T
-synuclein-expressing cells, basal conditions; c,
untransfected cells,
FeCl2-H2O2;
d, A53T -synuclein-expressing cells,
FeCl2-H2O2. Under basal
conditions, none of the cells showed staining for iron, but after
treatment with 10 mM FeCl2 and 100 µM H2O2 for 48 hr, the A53T cells
showed much more iron reactivity (blue, iron stain;
pink, nuclear-cytoplasmic counterstain).
|
|
| |
DISCUSSION |
-Synuclein has gained prominence because mutations in
-synuclein cause familial PD in several kindreds and because the
protein accumulates in Lewy bodies (Goedert et al., 1999). In
vitro studies using recombinant -synuclein show that the
protein has a strong tendency to aggregate and that the A53T and A30P
mutations increase the rate of aggregation (Conway et al., 1998;
Hashimoto et al., 1998; Giasson et al., 1999). We have now shown that
-synuclein also forms aggregates in neurons when cells are exposed
to iron or iron plus either hydrogen peroxide or dopamine, which
generate free radicals. Moreover, we observe that the A53T
-synuclein mutation shows an increased tendency to form aggregates,
which is consistent with observations in vitro (Conway et
al., 1998, 2000; Hashimoto et al., 1998; Giasson et al., 1999). The
aggregates also contain ubiquitin, which is known to be a major
component of Lewy bodies. Equally striking is the fact that aggregation is largely restricted to -synuclein, as shown by the observation that actin does not aggregate under these conditions. Cell toxicity per
se does not appear to cause aggregation because treatment with dopamine
alone, which causes toxicity, does not induce -synuclein aggregation. On the other hand, treating cells with iron does induce
-synuclein aggregation, and overexpressing -synuclein sensitizes
cells to iron-mediated toxicity. Thus, there appears to be a linkage
between toxicity, aggregation, iron, and -synuclein. We propose that
aggregation of -synuclein increases cellular iron content, which
increases oxidative toxicity. Thus, the interaction between
-synuclein and iron might be a critical, and causal, step in the
pathophysiology of Parkinson's disease.
Our results implicate iron in the pathophysiology of -synuclein.
Exposure of the cells to iron plus other free radical generators, such
as dopamine or hydrogen peroxide, was able to induce -synuclein aggregation in all of the cell lines. On the other hand, exposure of
cells to dopamine alone did not induce aggregation. These results point
to the important role that iron plays in inducing -synuclein aggregation. In the presence of iron, concentrations of dopamine as low
as 5 µM induce aggregation of all forms of -synuclein (wild-type, A30P, and A53T), which is within the range of dopamine that
is thought to be present in the neurons of the substantia nigra.
There is a large amount of circumstantial evidence implicating
oxidative factors, especially iron, in both the etiology and pathogenesis of PD and other diseases that exhibit aggregated -synuclein, such as multiple systems atrophy (Koga et al., 1998; Martin et al., 1998; Dickson et al., 1999). Because iron-induced -synuclein aggregation is dose-dependent, the accumulation of iron
in the substantia nigra that occurs during aging or Lewy body disease
might increase the rate of aggregation of -synuclein. In fact, iron
has been shown recently to be present in Lewy bodies in the substantia
nigra (Castellani et al., 2000). The presence of dopamine combined with
the accumulation of iron substantia nigra of elderly subjects might
therefore promote the aggregation of -synuclein and formation of
Lewy bodies.
Lewy bodies also contain a large amount of ubiquitin (Leroy et al.,
1998). Our studies revealed that the aggregates formed in the BE-M17
cells in response to the conditions that produced -synuclein
aggregation also contain ubiquitin. The amount of ubiquitin protein
that accumulated in the aggregates appeared to be proportional to the
amount of -synuclein aggregation. This suggests that the process of
-synuclein aggregation is coupled to ubiquitination. Many studies
have observed that both -synuclein and ubiquitin are abundant in
Lewy bodies and other types of -synuclein pathology (Spillantini et
al., 1997, 1998; Masliah et al., 2000). Whether ubiquitin or
-synuclein is more abundant in iron-induced aggregates was not
determined in our experiments because the amount of antigen detected
varied depending on the method and antibody used. Anti-ubiquitin
antibodies were more sensitive than anti--synuclein antibodies by
immunoblot, whereas the converse was true by immunocytochemistry. Similarly, antibodies to -synuclein also displayed differential sensitivities, depending on the epitope. The monoclonal antibody directed against the N terminus was better than the SC1 antibody in
detecting aggregates by immunoblotting, whereas the SC1 antibody was
better than the monoclonal antibody at detecting aggregates by
immunocytochemistry. The differences in sensitivity might depend on
epitope availability and have been noted by other investigators (Serpell et al., 2000). The most parsimonious conclusion from this data
is that -synuclein and ubiquitin are both abundant in the aggregates.
One of the important questions regarding -synuclein aggregation and
Lewy body formation is whether these processes harm the cell. Lewy
bodies could be inert tombstone markers that occur in response to free
radical damage, or they might be toxic agents that harm the cell.
Examples of both situations exist in the literature. Aggregated A is
toxic to neurons, whereas the lipofuscin appears to be innocuous to
cells (Behl et al., 1994). The Huntingtin protein presents an
intermediate situation in which the toxicity associated with Huntingtin
appears to precede aggregation, and aggregation of Huntingtin
might even be protective (Saudou et al., 1998). Our previous studies
showed that transient overexpression of -synuclein is toxic to a
variety of cells, including two neuronal cell lines, SK-NSH and
PC12 (Ostrerova et al., 1999). Consistent with this observation,
Masliah et al. (2000) have shown recently that mice overexpressing
-synuclein show an age-related loss of dopaminergic terminals and
motor impairment, which could be indicative of toxicity. In the present
study, we observed that -synuclein increases iron-mediated toxicity
and that in some assays the A53T -synuclein construct was more toxic
than the wild-type or A30P -synuclein constructs. The amount of
toxicity induced by each construct generally reflects the tendency to
induce aggregation. These data suggest that -synuclein can be
harmful to neurons under conditions that induce its aggregation.
Recent studies on transgenic animals also suggest that aggregation of
-synuclein is harmful to neurons. Masliah et al. (2000) noted
dopaminergic dysfunction in transgenic mice expressing wild-type human
-synuclein. Even stronger effects were observed in
Drosophila overexpressing -synuclein. Feany and Bender
(2000) observed dopaminergic dysfunction and dopaminergic neuronal
death associated with development of -synuclein aggregates. Thus,
increasing evidence suggests that neurons with dopamine develop
-synuclein aggregates and degenerate as these aggregates develop.
The potential significance of these data is clear. Although the
deposits we report here are not Lewy bodies, it is remarkable that they
resemble these structures in many of their apparent constituents and
are more prone to form in cells bearing pathogenic mutations. This
suggests that they may be imperfect markers of the same processes that
lead to Lewy bodies in vivo. The data we present here link
familial mutations in -synuclein with iron and aggregation and
suggest that the pathogenic process in PD involves the pushing of
-synuclein protein across a threshold into cellular aggregates,
which are, directly or indirectly, toxic to the cells that contain
them. Pathogenic -synuclein mutations appear to lower that threshold
in vitro and, as we show here, -synuclein overexpression
and oxidative stress can also both push transfected cells over this
threshold. From a therapeutic standpoint, these data suggest that
antioxidants and agents that lead to reduction of -synuclein
expression are legitimate targets for therapy and that other causes of
PD, whether genetic or environmental, will directly interact with
-synuclein metabolism.
| |
FOOTNOTES |
Received Feb. 22, 2000; revised June 5, 2000; accepted June 5, 2000.
This work was supported by a grant from the Retirement Research
Foundation (to B.W.) and the National Parkinson Foundation (to
B.W.).
Correspondence should be addressed to Dr. Benjamin Wolozin, Department
of Pharmacology, Loyola University Medical Center, Building 102, Room
4644, 2160 South 1st Avenue, Maywood, IL 60154. E-mail:
bwolozi{at}luc.edu.
| |
REFERENCES |
-
Behl C,
Davis J,
Lesley R,
Schubert D
(1994)
Hydrogen peroxide mediates amyloid protein toxicity.
Cell
77:817-827[ISI][Medline].
-
Castellani R, Siedlak S, Perry G, Smith
M (2000) Sequestration of iron by Lewy bodies in Parkinson's
disease. Acta Neuropathologica, in press.
-
Chiueh C,
Miyake H,
Peng M
(1993)
Role of dopamine autoxidation, hydroxyl radical generation, and calcium overload in underlying mechanisms involved in MPTP-induced parkinsonism.
Adv Neurol
60:251-258[Medline].
-
Conway K,
Harper J,
Lansbury P
(1998)
Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease.
Nat Med
4:1318-1320[ISI][Medline].
-
Conway KA,
Lee SJ,
Rochet JC,
Ding TT,
Williamson RE,
Lansbury Jr PT
(2000)
Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy.
Proc Natl Acad Sci USA
97:571-576[Abstract/Free Full Text].
-
Dickson D,
Lin W-L,
Liu W,
Yen S
(1999)
Multiple system atrophy: a sporadic synucleinopathy.
Brain Pathol
9:721-732[ISI][Medline].
-
Feany MB,
Bender WW
(2000)
A Drosophila model of Parkinson's disease.
Nature
404:394-398[Medline].
-
Giasson B,
Uryu K,
Trojanowski J,
Lee V
(1999)
Mutant and wild type human -synucleins assemble into elongated filaments with distinct morphologies in vitro.
J Biol Chem
274:7619-7622[Abstract/Free Full Text].
-
Gibb W,
Lees A
(1988)
The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease.
J Neurol Neurosurg Psychiatry
51:745-752[Abstract].
-
Goedert M,
Spillantini M,
Davies S
(1999)
Filamentous nerve cell inclusions in neurodegenerative diseases.
Curr Opin Neurobiol
8:619-632.
-
Hashimoto M,
Hsu L,
Sisk A,
Xia Y,
Takeda A,
Sundsmo M,
Masliah E
(1998)
Human recombinant NACP/-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease.
Brain Res
799:301-306[ISI][Medline].
-
Hashimoto M,
Hsu LJ,
Xia Y,
Takeda A,
Sisk A,
Sundsmo M,
Masliah E
(1999)
Oxidative stress induces amyloid-like aggregate formation of NACP/-synuclein in vitro.
NeuroReport
10:717-721[ISI][Medline].
-
Jenner P,
Olanow C
(1998)
Understanding cell death in Parkinson's disease.
Ann Neurol
44:S72-S84[ISI][Medline].
-
Koga H,
Kondo A,
Kimura T,
Takamatsu J
(1998)
Familial multiple system degeneration associated with iron deposition: determination of subcellular localization of elementary iron by energy-dispersive X-ray analysis.
Clin Neuropathol
17:35-40[Medline].
-
Leroy E,
Boyer R,
Auburger G,
Leube B,
Ulm G,
Mezey E,
Harta G,
Brownstein M,
Jonnalagada S,
Chernova T,
Dehejia A,
Lavedan C,
Gasser T,
Steinbach P,
Wilkinson K,
Polymeropoulos M
(1998)
The ubiquitin pathway in Parkinson's disease.
Nature
395:451-452[Medline].
-
Markopoulou K,
Wszolek Z,
Pfeiffer R,
Chase B
(1999)
Reduced expression of the G209A -synuclein allele in familial parkinsonism.
Ann Neurol
46:374-381[ISI][Medline].
-
Martin WR,
Roberts TE,
Ye FQ,
Allen PS
(1998)
Increased basal ganglia iron in striatonigral degeneration: in vivo estimation with magnetic resonance.
Can J Neurol Sci
25:44-47[ISI][Medline].
-
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].
-
Ostrerova N,
Petrucelli L,
Farrer M,
Mehta N,
Alexander P,
Choi P,
Palacino J,
Hardy J,
Wolozin B
(1999)
-Synuclein shares physical and functional homology with 14-3-3 proteins.
J Neurosci
19:5782-5791[Abstract/Free Full Text].
-
Paik S,
Shin H,
Lee J,
Chang C,
Kim J
(1999)
Copper(II)-induced self-oligomerization of -synuclein.
Biochem J
340:821-828.
-
Papadimitriou A,
Veletza V,
Hadjigeorgiou G,
Patrikiou A,
Hirano M,
Anastasopoulos I
(1999)
Mutated alpha-synuclein gene in two Greek kindreds with familial PD: incomplete penetrance?
Neurology
52:651-654[Abstract/Free Full Text].
-
Polymeropoulos M,
Higgins J,
Golbe L,
Johnson W,
Ide S,
Di Iorio G,
Sanges G,
Stenroos E,
Pho L,
Schaffer A,
Lazzarini A,
Nussbaum R,
Duvoisin R
(1997)
Mapping of a gene for Parkinson's disease to chromosome 4q21-q23.
Science
276:1197-1199[Free Full Text].
-
Saudou F,
Finkbeiner S,
Devys D,
Greenberg M
(1998)
Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with formation of intranuclear inclusions.
Cell
95:55-66[ISI][Medline].
-
Serpell LC,
Berriman J,
Jakes R,
Goedert M,
Crowther RA
(2000)
Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation.
Proc Natl Acad Sci USA
97:4897-4902[Abstract/Free Full Text].
-
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].
-
Tanner C,
Ottman R,
Goldman S,
Ellenberg J,
Chan P,
Mayeux R,
Langston J
(1999)
Parkinson disease in twins: an etiologic study.
JAMA
281:341-346[Abstract/Free Full Text].
-
Wolozin B,
Behl C
(2000)
Mechanisms of neurodegenerative disorders. I. Protein aggregates.
Arch Neurol
57:793-796[Free Full Text].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20166048-07$05.00/0
This article has been cited by other articles:
|
|
|
|
 
M. Kostka, T. Hogen, K. M. Danzer, J. Levin, M. Habeck, A. Wirth, R. Wagner, C. G. Glabe, S. Finger, U. Heinzelmann, et al.
Single Particle Characterization of Iron-induced Pore-forming {alpha}-Synuclein Oligomers
J. Biol. Chem.,
April 18, 2008;
283(16):
10992 - 11003.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Zhu, W. Xie, T. Pan, P. Xu, M. Fridkin, H. Zheng, J. Jankovic, M. B. H. Youdim, and W. Le
Prevention and restoration of lactacystin-induced nigrostriatal dopamine neuron degeneration by novel brain-permeable iron chelators
FASEB J,
December 1, 2007;
21(14):
3835 - 3844.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Basu, M. L. Mohan, X. Luo, B. Kundu, Q. Kong, and N. Singh
Modulation of Proteinase K-resistant Prion Protein in Cells and Infectious Brain Homogenate by Redox Iron: Implications for Prion Replication and Disease Pathogenesis
Mol. Biol. Cell,
September 1, 2007;
18(9):
3302 - 3312.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. E. Oakley, J. F. Collingwood, J. Dobson, G. Love, H. R. Perrott, J. A. Edwardson, M. Elstner, and C. M. Morris
Individual dopaminergic neurons show raised iron levels in Parkinson disease
Neurology,
May 22, 2007;
68(21):
1820 - 1825.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. da Costa, J. Dunys, F. Brau, S. Wilk, R. Cappai, and F. Checler
6-Hydroxydopamine but Not 1-Methyl-4-phenylpyridinium Abolishes {alpha}-Synuclein Anti-apoptotic Phenotype by Inhibiting Its Proteasomal Degradation and by Promoting Its Aggregation
J. Biol. Chem.,
April 7, 2006;
281(14):
9824 - 9831.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
