From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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A link between Since it is unclear how Expression and Purification of Western Blotting--
Proteins were resolved on slab gels by
SDS-polyacrylamide gel electrophoresis (PAGE) (26) and
electrophoretically transferred onto nitrocellulose membranes
(Schleicher and Schuell) in buffer containing 48 mM Tris,
39 mM glycine, and 10% methanol. Membranes were blocked
with a 1% solution of powdered skim milk dissolved in Tris buffered
saline-Tween (50 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Tween 20), incubated with anti- Centrifugal Sedimentation--
Following incubation at 37 °C
in 100 mM sodium acetate, pH 7.0, with continuous shaking,
samples were centrifuged at 150,000 × g for 30 min,
SDS-sample buffer (10 mM Tris, pH 6.8, 1 mM
EDTA, 40 mM dithiothreitol, 1% SDS, 10% sucrose) was
added to pellets and supernatants and heated to 100 °C for 15 min.
Antibody Generation--
SNL-1 and SNL-4 are affinity-purified
rabbit polyclonal antibodies raised against peptides corresponding to
amino acid residues 104-119 and 2-12 in Immunoelectron Microscopy and Negative
Staining--
The purity of recombinant -Synuclein is a soluble presynaptic protein
which is pathologically redistributed within intracellular lesions
characteristic of several neurodegenerative diseases. Here we
demonstrate that wild type and two mutant forms of
-synuclein linked
to familial Parkinson's disease (Ala30
Pro and
Ala53
Thr) self-aggregate and assemble into
10-19-nm-wide filaments with distinct morphologies under defined
in vitro conditions. Immunogold labeling demonstrates that
the central region of all these filaments are more robustly labeled
than the N-terminal or C-terminal regions, suggesting that the latter
regions are buried within the filaments. Since in vitro
generated
-synuclein filaments resemble the major ultrastructural
elements of authentic Lewy bodies that are hallmark lesions of
Parkinson's disease, we propose that self-aggregating
-synuclein is
the major subunit protein of these filamentous lesions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-Synuclein is a small, 140-amino acid protein characterized by
acidic stretches toward the C terminus and six repetitive, degenerate
amino acid sequences of the prototype KTKEGV between amino acid
residues 10 and 86 (1, 2). The first synuclein identified and cloned
was from the electric eel Torpedo california (3). In the
latter study, the authors also cloned a rat synuclein homologue that
was later termed
-synuclein (4).
-Synuclein was first associated
with a human neurodegenerative disease when a 35-amino acid peptide
corresponding to residues 61-95 in
-synuclein was purified from
Alzheimer's disease senile plaques (5). This peptide was referred to
as the non-amyloid component of senile plaques
(NAC)1 and its precursor
protein, i.e.
-synuclein, was designated NACP (5). The
zebra finch homologue of
-synuclein, synelfin, was identified as a
gene product potentially implicated in neuronal plasticity (6).
-Synuclein is predominantly a neuronal protein expressed in brain
and localized in axon terminals (5-8). Because
-synuclein is in
close proximity to and is loosely associated with presynaptic vesicles,
it may modulate the function of synaptic vesicles (6, 7). Although
-synuclein has very little structure in aqueous solution (9), it
associates with small unilamellar acidic phospholipid vesicles in
vitro and acquires increased
-helicity (10). Thus, it has been
proposed that
-synuclein may bind vesicles by forming amphipathic
helices similar to apolipoproteins (6).
-synuclein and Parkinson's disease (PD) first
surfaced when Polymeropoulos et al. (11) reported a missense mutation (Ala53
Thr) in
-synuclein in four Italian
and Greek kindreds with autosomal dominant PD. This finding was
followed by the demonstration of
-synuclein immunoreactivity in Lewy
bodies (LBs) and Lewy neurites in patients with sporadic PD and
dementia with LBs (DLB) (12). Significantly, these lesions are
pathological hallmarks of PD and DLB (13, 14). Subsequent studies
confirmed that normal, truncated, and aggregated
-synuclein are
major components of LBs and Lewy neurites and that antibodies to
-synuclein are the most reliable and consistent immunological probes
for detecting these lesions in situ (15-19). More recently,
-synuclein was also shown to be a prominent component of glial cell
inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs) that are
characteristic of multisystem atrophy (MSA) and Hallervorden-Spatz
disease (20-24). Furthermore, a second pathogenic mutation in
-synuclein (Ala30
Pro) also was reported in another
familial PD kindred (25).
-synuclein, a very soluble protein, ends up
in cellular inclusions, we carried out studies to determine whether
wild type (WT) and/or mutants of
-synuclein can self-aggregate in vitro. Here, we report that the SDS solubility of wild
type and mutant
-synucleins is reduced after incubation in aqueous solution at physiological temperature and this change in physical property was attributed to the formation of elongated filaments.
EXPERIMENTAL PROCEDURES
-Synuclein--
Human WT,
A30P, and A53T
-synuclein cDNAs subcloned into the bacterial
expression vector pRK172 were expressed in Escherichia coli
BL21 (DE3). Bacterial pellets were resuspended in high-salt lysis
buffer (0.75 M NaCl, 100 mM MES, pH 7.0, 1 mM EDTA) containing a mixture of protease inhibitors,
heated to 100 °C for 10 min, and centrifuged at 70,000 × g for 30 min. The supernatants were dialyzed against 10 mM Tris, pH 7.5, applied to a Mono Q column, and eluted
with a 0-0.5 M NaCl gradient. Protein concentration was
determined using the bicinchoninic acid protein assay (Pierce) and
bovine serum albumin as a standard.
-synuclein antibody LB509 (15), followed with a goat anti-mouse IgG horseradish
peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories,
Inc.) and developed with 3,3'-diaminobenzidine.
-Synuclein was resolved on SDS-PAGE, stained with Coomassie
Brilliant Blue R-250, and quantified by densitometry.
-synuclein, respectively.
Syn 202, Syn 204, Syn 205 and Syn 208 are novel mouse monoclonal
antibodies raised to synucleins, as described previously (20). The
epitopes for Syn 202 and 205 are localized to amino acid residues
130-140, while the Syn 204 and 208 epitopes map to residues 87-110.
The relative activities of the antibodies were determined by
enzyme-linked immunosorbent assay using
-synuclein as the antigen
and by performing serial dilutions of the antibodies.
-Synuclein filaments were decorated with
anti-
-synuclein antibodies and negative stained with uranyl acetate
as described previously (27). Briefly, assembled
-synuclein
filaments were absorbed to 300 mesh carbon coated copper grids and
stained with 1% uranyl acetate or labeled with antibodies to
-synuclein followed by secondary antibodies conjugated to 10 nm gold
and staining with 1% uranyl acetate.
RESULTS
-synuclein proteins was demonstrated
in Coomassie Blue-stained SDS-PAGE gels (Fig.
1A). No contaminating proteins
were seen even when 100 µg of purified recombinant
-synucleins were loaded in separate lanes of an SDS-PAGE gel (data not shown). Incubation of 5 mg/ml WT
-synuclein at 37 °C for 48 h in a
number of different buffers resulted in the aggregation of
-synuclein as reflected by decreased mobility on SDS-PAGE (Fig.
1B). Diffuse smears of the incubated proteins on SDS-PAGE
gels may reflect the rapid association/dissociation of
-synuclein in
the presence of SDS. Temperature was a major determinant of
-synuclein aggregation, since under the same conditions, incubation
at 37 °C generated abundant aggregation that was not significantly
detected when the protein was incubated at room temperature (Fig.
1B). WT (Fig. 1C), A30P (Fig. 1D), and
A53T (Fig. 1E)
-synucleins demonstrated a similar ability
to aggregate, although A53T
-synuclein seemed to aggregate to a
slightly greater extent at lower concentrations. The ability of
-synuclein to polymerize was confirmed with centrifugal sedimentation experiments (Fig. 2). WT,
A30P, and A53T
-synuclein polymerization was concentration- and
time-dependent, and the A53T mutant had a greater
propensity to polymerize.
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Fig. 1.
Aggregation of WT, A30P, and A53T
-synuclein as demonstrated by SDS-PAGE followed by
Western blotting. Anti-
-synuclein mouse monoclonal antibody LB
509 (15) was used to detect
-synuclein by Western blotting.
A, 10 µg of purified human WT, A30P, and A53T
-synucleins were resolved on SDS-PAGE and stained with Coomassie
Brilliant Blue R-250. B, Western blot of 5 mg/ml WT
-synuclein non-incubated (N) or incubated in
phosphate-buffered saline (137 mM NaCl, 2.7 mM
KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.0)
(PBS), 10 mM Tris, pH 7.0 (T), 10 mM Tris, pH 7.0, 100 mM NaCl (TN),
100 mM Tris, pH 7.0 (HT), 100 mM
MES, pH 7.0 (M), or 100 mM sodium acetate, pH
7.0 (Ac) for 48 h at room temperature or at 37 °C
(') with constant shaking. Following incubation, SDS-sample buffer was
added, and samples were loaded on a 12% polyacrylamide gel.
C-E, Western blot analysis of WT (C), A30P
(D), and A53T (E)
-synucleins at increasing
concentrations after incubation for 48 h at 37 °C in 100 mM sodium acetate, pH 7.0, with continuous shaking. WT or
mutants of
-synuclein (5 mg/ml) that were not incubated
(N) also were also loaded on each respective gel. In
B-E, 1 µl of each sample was loaded in separate lanes of
the gels. Similar results were obtained in two independent
experiments. The molecular masses of markers in kilodaltons are
indicated on the left of each gel.
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Fig. 2.
-Synuclein polymerization is
time- and concentration-dependent. WT, A30P, and A53T
-synucleins were incubated at 37 °C in 100 mM sodium
acetate, pH 7.0, for 0-48 h. The proteins were sedimented as described
under "Experimental Procedures," and the percentage of each protein
in the pellet is indicated on the y axis. The protein
concentrations used for each incubation are indicated at the
bottom of each bar graph; n = 2. The range for each set of experiments was less than ±13% from
the mean.
Electron microscopic analysis of wild type and mutant -synucleins
postincubation revealed that they formed elongated filaments that
frequently attained lengths of several microns (Fig.
3). In some fields, a plethora of
-synuclein filaments filled the whole area on the grid (Fig.
3A). Interestingly, the morphology of the different
synuclein filaments varied. For example, WT
-synuclein mainly formed
straight filaments, although twisted filaments were also observed (Fig.
3B). In contrast, A53T
-synuclein predominantly formed
twisted filaments that appeared to contain two protofilaments in a
regular helical fibril (Fig. 3C), while A30P
-synuclein formed filaments that were straight (Fig. 3D). WT and A30P
-synuclein filaments had diameters ranging between 10 and 15 nm
(mean = 12 ± 1.4 nm) and 11-16 nm (mean = 13 ± 1.4 nm), respectively, whereas A53T
-synuclein filaments were
slightly wider with widths ranging between 16 and 19 nm (mean = 17 ± 1.1 nm).
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-Synuclein filaments were only modestly labeled with antibodies to
the N terminus (SNL-4) (Fig.
4A) or the C-terminal region (SNL-1; Syn 202 and Syn 205) (Fig. 4, B and C;
data not shown) of
-synuclein. However, antibodies to epitopes
within the central part of
-synuclein (Syn 204 and Syn 208)
demonstrated very strong labeling (Fig. 4, D-F). Antibodies
SNL-1, SNL-4, Syn 202, and Syn 205 were used at 10-20-fold higher
relative immunoreactiveactivity than antibodies Syn 204 and Syn 208. Thus, these results may suggest that the N- and C-terminal regions of
-synuclein are less accessible than the central region within these
filaments, and this may imply that both ends of the polypeptide are
involved in polymer formation and embedded within the
filaments.
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DISCUSSION |
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Despite the fact that -synuclein is a small soluble synaptic
protein that is largely devoid of secondary structure in aqueous buffer, we show here that mutant and WT recombinant
-synucleins polymerize into morphologically distinct filaments under a variety of
in vitro conditions. Furthermore,
-synuclein
polymerization is temperature-, concentration-, and
time-dependent, and the protein subunits are
topographically organized within these polymers as reflected by the
paucity of immunoreactivity for antibodies to
-synuclein epitopes at
both ends of the polypeptide relative to those in the central region.
Although a recent report demonstrated that wild-type
-synuclein
forms Thioflavin-S-reactive aggregates and filaments, especially at
elevated temperature (28), our studies substantially extend these
preliminary observations by comparing the morphology of filaments
formed from WT and mutant
-synucleins as well as the topography of
-synuclein in these filaments. A possible model consistent with
these observations is that
-synuclein exists in numerous
conformational states in aqueous solution, but when molecules with
conformations compatible with dimerization interact, they may stabilize
each other in this dimerized conformation, and these dimers may then
serve as seeds for polymerization.
The findings presented here and in previous reports suggest that
-synuclein is the major building block of the filaments that form
LBs. First, anti-
-synuclein antibodies stain LBs more intensely and
consistently than any other antibodies, including anti-ubiquitin
antibodies (12, 15-17, 19). Second, anti-
-synuclein immunoreactivity also is abundant in pale bodies (19, 24), which are
believed to be precursor lesions of LBs. Third, neurofilaments, another
filamentous component of LBs (29-31), are detected only in a small
percentage of pale bodies (32), and most anti-neurofilament antibodies
only label a subset of LBs (30). However, at least one
anti-neurofilament antibody that is specific for the mid-size neurofilament subunit (RMO32) labels nearly all cortical LBs (30). Fourth, the filaments generated in vitro from WT and mutant
-synucleins are morphologically similar to the LB filaments
visualized in situ (30, 31, 33) as well as to synuclein
filaments recovered from Sarkosyl-insoluble preparations of DLB brains
(16), while normal neurofilaments have side arms (27, 34) that are not seen in LB fibrils (31, 33).
LB filament formation in vivo may result from -synuclein
accumulation, and this may be due to a reduction in the fast axonal transport (35) or overexpression of
-synuclein. Furthermore, once
-synuclein reaches a critical intracellular concentration, it may
polymerize into filaments that aggregate into LBs or LB precursors
which entrap other cytoplasmic components, and similar mechanisms may
lead to the formation of Lewy neurites. Additionally, the reduced
ability of A30P
-synuclein to bind vesicles (35) may play a
mechanistic role in the onset/progression of some forms of familial PD,
while the A53T mutation may be pathogenic because it increases the
propensity of
-synuclein to polymerize into LB filaments (see Figs.
1, C-E, and 2). The accelerated formation of filaments as a
consequence of the A53T mutation has also been observed in a recent
publication by Conway et al. (42). Interestingly, Crowther
et al. (43) have reported that carboxyl-terminally truncated
-synuclein may be more prone to form filaments than the full-length
protein. Although the pathological implications of the latter finding
is still unclear, it is possible that aberrant proteolysis of
-synuclein may create a pool of these shorter products that may play
a role in initiating
-synuclein filament assembly.
Although -synuclein has been reported to be expressed predominantly
in neurons, it also is a major component of GCIs and NCIs in MSA brains
(20-24). Ultrastructurally, GCIs and NCIs have been reported to be
composed of 15-40-nm filaments (23, 36-41), but some GCIs appear to
contain tubular filaments (38, 41), although this issue remains
controversial (36). Recently, the filaments in GCIs have been shown to
be intensely labeled with anti-
-synuclein antibodies at the
ultrastructural level (15, 23), and Sarkosyl-insoluble
-synuclein filaments with straight and twisted morphologies similar
to our synthetic filaments also have been observed in extracts of MSA
brains (21). Perhaps the tubular appearance of some GCI fibrils is due
to the incorporation of other proteins into
-synuclein filaments or
these tubular fibrils may reflect the presence of microtubules trapped
within bundles of
-synuclein filaments. This uncertainty
notwithstanding, we speculate that the anomalous expression,
accumulation, and subsequent polymerization of
-synuclein lead to
the formation of GCIs in MSA brain. However, future research will be
needed to determine whether
-synuclein polymers serve as a
proteinaceous trap and if
-synuclein co-assembles with other
proteins previously co-localized in LBs and GCIs in situ.
Finally, efforts to over express WT and mutant
-synucleins in
vivo will help to elucidate the role of
-synuclein filaments in
the pathogenesis of PD, DLB, MSA, and related
-synucleinopathies.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Goedert for the -synuclein
cDNA constructs and epitope localization; Drs. B. Balin, D. Murphy,
and P. Sterling for help with electron microscopy; and Dr. S. Rueter
for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by grants from the National Institute on Aging and a Pioneer Award from the Alzheimer's Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Human Frontier Science Program Organization.
§ To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, Maloney 3, HUP, Philadelphia, PA 19104-4283; Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee{at}mail.med.upenn.edu.
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ABBREVIATIONS |
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The abbreviations used are: NAC, non-amyloid component of senile plaques; NACP, NAC precursor protein; DLB, dementia with Lewy bodies; GCI, glial cell inclusion; LB, Lewy body; MSA, multisystem atrophy; NCI, neuronal cytoplasmic inclusion; PAGE, polyacrylamide gel electrophoresis; PD, Parkinson's disease; WT, wild type; MES, 2-[N-morpholino]ethanesulfonic acid.
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REFERENCES |
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