Both Familial Parkinson's Disease Mutations Accelerate
-Synuclein Aggregation*
Linda
Narhi
,
Stephen J.
Wood
,
Shirley
Steavenson
,
Yijia
Jiang,
Gay May
Wu,
Dan
Anafi,
Stephen A.
Kaufman,
Francis
Martin,
Karen
Sitney,
Paul
Denis,
Jean-Claude
Louis,
Jette
Wypych,
Anja Leona
Biere, and
Martin
Citron§
Amgen, Inc., Thousand Oaks, California 91320-1789
 |
ABSTRACT |
Parkinson's disease (PD) is a neurodegenerative
disorder that is pathologically characterized by the presence of
intracytoplasmic Lewy bodies, the major component of which are
filaments consisting of
-synuclein. Two recently identified point
mutations in
-synuclein are the only known genetic causes of PD, but
their pathogenic mechanism is not understood.
Here we show that both wild type and mutant
-synuclein form
insoluble fibrillar aggregates with antiparallel
-sheet structure upon incubation at physiological temperature in vitro.
Importantly, aggregate formation is accelerated by both PD-linked
mutations. Under the experimental conditions, the lag time for the
formation of precipitable aggregates is about 280 h for the wild
type protein, 180 h for the A30P mutant, and only 100 h for
the A53T mutant protein. These data suggest that the formation of
-synuclein aggregates could be a critical step in PD pathogenesis,
which is accelerated by the PD-linked mutations.
 |
INTRODUCTION |
Parkinson's disease is a neurodegenerative disorder that
predominantly affects dopaminergic neurons in the nigrostriatal system but also several other regions of the brain. Two dominant mutations, A53T and A30P, in
-synuclein cause familial early onset PD (1, 2).
The function of
-synuclein and the pathogenic mechanism of these
mutations is unknown, but
-synuclein has been detected in Lewy
bodies (3-5) and shown to be their major filamentous component (6).
Lewy bodies are a pathological hallmark of PD (7-9), and we therefore
hypothesized that the PD mutations would cause or enhance
-synuclein
aggregation. Indeed, a very recent publication demonstrated in
vitro fibrillization of A53T mutant but not A30P mutant or wild
type
-synuclein (10). Here we demonstrate aggregation of all forms
of
-synuclein. In a complete aggregation time course, we show that
there is an aggregation continuum; although all forms of
-synuclein
do aggregate, aggregation is accelerated for both mutants; A30P
aggregates slightly faster than wild type, and A53T aggregates much
faster. Because both mutant forms enhance the aggregation tendency
observed in the wild type, we hypothesize that aggregation of
-synuclein may be important in all forms of
PD.1
 |
EXPERIMENTAL PROCEDURES |
Cloning, Bacterial Expression, and Purification of
-Synuclein--
A 536-bp human
-synuclein cDNA was obtained
by polymerase chain reaction amplification from an adult human brain
cDNA library using primers corresponding to nucleotides 20-42 and
532-556 of the published sequence (11). Polymerase chain
reaction-based site-directed mutagenesis of this sequence was used to
generate the mutant forms A53T/ A30P, and A53T + A30P. For bacterial
expression, all 4 forms were amplified using the primers
TGTGGTCTAGAAGGAGGAATAACATATGGATGTATTCATGAAAGGTCTGTCAAAGGCCAAGGAGGGTGTTGTG and GGGACCGCGGCTCGAGATTAGGCTTCAGGTTCGTAGTCTTGATAACCTTCCTCA to alter 3 codons near the 5' end and 1 codon near the 3' end to more highly
utilized Escherichia coli codons. The resulting PCR products
were digested with NdeI and XhoI and cloned into
the E. coli expression vector pAMG21 (12). The correct DNA
sequence of all four constructs was confirmed by DNA
sequencing. E. coli containing the various plasmids were
induced during fermentation. E. coli cell paste was
homogenized in 20 mM Tris, 100 mM NaCl, pH 7.5, with protease inhibitor mixture Complete (Boehringer Mannheim). Cells
in suspension were broken by passaging through a Microfluidizer, and a
clarified lysate supernatant was collected after centrifugation at
18,000 × g for 45 min. E. coli
contaminating proteins were precipitated by acid precipitation of the
lysate supernatant. The pH was adjusted to 3.5, and after stirring for
20 min, the mixture was centrifuged for 1 h at 27,000 × g. After adjusting the pH of the resulting supernatant to
7.5, the sample was applied to Q-Sepharose FF (Amersham Pharmacia
Biotech), equilibrated in 20 mM Tris, pH 7.5, and eluted
with a NaCl gradient in equilibration buffer.
-Synuclein-containing
fractions were identified by SDS-polyacrylamide gel electrophoresis and
are >99% pure. The concentration of
-synuclein was determined by
measuring absorbance at 280 nm and employing
2800.1% of 0.354, determined by
using Genetics Computer Group software.
Aggregation of
-Synuclein--
Purified
-synuclein samples
were concentrated to 7 mg/ml using Centricon-3 spin filters (Amicon).
After concentration, the samples were centrifuged for 10 min at
100,000 × g to remove any aggregates that could have
formed during the concentration step. The supernatants were all
adjusted to a final concentration of 7 mg/ml using Tris-buffered
saline, which consists of 20 mM Tris, pH 7.5, and 0.2 M NaCl. The samples were then dispensed into 1.5 ml of
Beckman ultracentrifuge microtubes and incubated at 37 °C. At
various time points, the samples were centrifuged at 100,000 × g for 10 min, and 11 µl of their supernatants were removed
and diluted to 110 µl with Tris-buffered saline. These dilutions were then analyzed by their absorbance at 280 nm. Finally, the remainder of
the incubations were vortexed for 30 s to resuspend pelleted material and allowed to continue incubating.
Circular Dichroism--
CD spectra were determined at 20 °C
on a Jasco J-715 Spectropolarimeter using water-jacketed cuvettes with
a path length of either 0.01 cm (for the far UV region, 250-190 nm,
secondary structure) or 1 cm (for the near UV region, 340-240 nm,
tertiary structure). Molar ellipticity was calculated using the protein
concentration determined as above and a mean residue weight of 103.
FTIR Measurement and Analysis--
FTIR spectra of aqueous
protein solutions and dried aggregates were recorded at 25 °C with a
Nicolet Magna 550 Fourier transform infared spectrometer equipped with
a deuterated triglycine sulfate detector. Protein solutions were
prepared for infared measurement in a sample cell (Spectra-Tech
FT04-036) that employed CaF2 windows separated by a 6-µm
spacer.
-Synuclein aggregates were centrifuged at 13000 rpm for 10 min, spread on a 3-M disposable IR card, and air-dried, and the
infrared spectra were recorded. The final protein spectrum was smoothed
with a 7-point Savitsky-Golay smooth function to remove the white
noise. Second derivative spectra were calculated with the derivative
function of the Nicolet Omnic software. To quantitate the secondary
structure from the second derivative spectra, the spectra were inverted
by multiplication by
1 and the curve fit (SpectraCalc Software from
Galactic Industries) with Gausian band profiles (13).
Atomic Force Microscopy--
Aggregated
-synuclein was
resuspended in phosphate-buffered saline, and this suspension was
vortexed for 10 s. 40 µl of this suspension was incubated on a
circular piece of mica for about 3 min. Excess liquid was removed, and
the sample on the mica was then imaged under 40 µl of
phosphate-buffered saline using a Digital Instruments Nanoscope III
atomic force microscope. The probe used for imaging was an
oxide-sharpened silicon nitride twin tip with a nominal spring constant
of 0.58 newton/m. The image was obtained in tapping mode in fluid using
a drive frequency of 8.67 kHz, a drive amplitude of 200 mV, and a set
point voltage of 0.252V.
Electron Microscopy--
Formvar-coated 300-mesh copper grids
were inverted over 20-µl drops of prepared
-synuclein aggregate
suspensions for 10 min. The grids were then rinsed in ultrapure water
to remove excess, nonadherent material and allowed to dry at room
temperature. The grids were placed sample down on 2% aqueous uranyl
acetate for 30 min, rinsed in water, and allowed to dry. The grids were
examined at 120 kV, and representative fields were photographed at
45,000 diameters' magnification on a Philips CM120 transmission
electron microscope.
 |
RESULTS |
To test the hypothesis that both PD mutations would cause or
enhance aggregation of
-synuclein and that this effect would be
detectable in vitro, we cloned human
-synuclein cDNA
(11) and generated bacterial expression constructs for the wild type protein, the two PD-linked variants, and a form containing both PD
mutations within the same molecule. After purification, all 4 proteins
run indistinguishably as a single band on SDS-polyacrylamide gel
electrophoresis at 19 kDa, and no differences in electrophoretic behavior were observed on native and nondenaturing gels (data not
shown). To address whether the wild type and the mutants differ in
their conformation, we performed CD and FTIR spectroscopy on solutions
of all four species. Fresh solutions of all four proteins showed the
natively unfolded structure previously described for the wild type
protein (14), with identical near and far UV CD spectra (Fig.
1). The FTIR spectra of these molecules
were also indistinguishable and indicated that they contain primarily
random coil structure. Thus, the initial conformation of the wild type
-synuclein and the mutants is identical.

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Fig. 1.
Fresh solutions of wild type and mutant
-synuclein exhibit the same natively unfolded
structure. Near UV CD spectra (A) and far UV CD spectra
(B) of wild type ( ), A30P (. . . . . .), A53T (.-.-),
and A30P/A53T (..-..-..-) -synuclein in phosphate-buffered
saline.
|
|
Because
-synuclein aggregation could be slow, we chose to analyze
solutions of
-synuclein over several days at three different temperatures. During the time frame of the experiment no aggregates formed when incubated at 4 °C or room temperature (data not shown). After incubation for several days at 37 °C, all four proteins began
to form insoluble aggregates that could be precipitated by
ultracentrifugation. This aggregation proceeded until most of the
material had fallen out of solution. This process could be further
accelerated by continuously shaking the solution (data not shown).
Importantly, in all experiments, aggregate formation was faster for the
PD-linked mutations than for the wild type, and the A53T mutation had a
more dramatic effect than the A30P mutation. This is most clearly seen
when the data are converted into approximate lag times before
precipitable aggregates are detected. The lag time for the wild type
protein was about 280 h, that of the A30P mutant was about
180 h, and that of the A53T mutant was only about 100 h. An
example of such an aggregation time course is shown in Fig.
2. At 11 distinct time points we separated the soluble and insoluble material using ultracentrifugation. The secondary structure of the protein that remained soluble was analyzed by CD and did not change for any of the four proteins throughout the course of the experiment (data not shown). The structure
of the protein that was in the pellet was analyzed by FTIR. As Fig.
3 clearly shows, aggregation and
precipitation of
-synuclein is accompanied by a dramatic change in
secondary structure, from the initial primarily random coil seen when
the
-synuclein is in solution structure (1650 cm
1
band, Fig. 3, top panel) to the final antiparallel
-sheet
structure present in the
-synuclein pellet (1629 cm
1,
Fig. 3, bottom panel). This structure is commonly observed
in protein aggregates. The spectra of the aggregate of all four forms are indistinguishable. To address whether the precipitates contain only
amorphous aggregates or distinct fibrillar structures, we analyzed them
by electron and atomic force microscopy (Fig.
4). Fibrils of the wild type and mutant
proteins were readily detected in the precipitates by electron
microscopy with positive staining. The diameter of the fibrils is
around 12 nm. Fluid phase atomic force microscopy clearly demonstrates
the presence of fibrils in their native aqueous environment.

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Fig. 2.
-Synuclein mutations show
accelerated aggregation. Aggregate formation of wild type and
mutant -synuclein solutions was analyzed by measuring the UV
absorption at 280 nm of protein in solution after ultracentrifugation.
Values are means ± S.E. of three different solutions.
Filled square, wild type; open diamonds, A53T;
open circles, A30P; open triangles,
A53T/A30P.
|
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Fig. 3.
Aggregate formation is accompanied by a
change in secondary structure from random coil to antiparallel
-sheet. The top panel is the second
derivative FTIR spectrum of the initial solution of wild type protein.
This spectrum shows primarily random coil when the -synuclein is in
its solution structure (1650 cm 1 band). The bottom
panel is the second derivative FTIR spectrum of the wild type
-synuclein aggregate. This spectrum shows the final antiparallel
-sheet structure present in the -synuclein pellet (1629 cm 1).
|
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Fig. 4.
Wild type and mutant
-synuclein form fibrils. A,
electron microscopy of wild type -synuclein aggregates demonstrates
the fibrillar nature of the precipitate. Fibers of various lengths are
observed in this representative field. The diameter of the fibrils is
around 12 nm. The bar represents 200 nm. B,
atomic force microscopy of A53T mutant -synuclein demonstrates the
presence of hydrated fibers in their native aqueous environment. The
image is a topographical height image of a 1-µm square area. Areas of
increasing brightness represent areas of increasing height. The
bar represents 200 nm.
|
|
 |
DISCUSSION |
Here we have shown that wild type and both mutant forms of
-synuclein can spontaneously form fibrillar
-sheet aggregates. The generation of such aggregates could be a critical step in the
formation of Lewy bodies, which contain full-length
-synuclein as
their major fibrillar component (6). Future studies will address in
more detail the kinetics of
-synuclein aggregation and fibril
morphology. The lag phase preceding
-synuclein fibril formation
(Fig. 2) could be indicative of a nucleation-dependent polymerization, and the fact that stirring or shaking dramatically increases the aggregation rate is also supportive of such a mechanism (for review, see Ref. 15). Interestingly, the
-synuclein mutants studied here all show a reduced lag phase with respect to wild type.
Under our conditions the wild type lag phase is about 280 h, that
of the A30P mutant is about 180 h, and that of the A53T mutant is
about 100 h. The actual concentration of
-synuclein within
cells forming Lewy bodies is unknown, but it is very likely to be much
lower than in this experimental setup. However, it is not unreasonable
to assume that in vivo, nuclei formation could be influenced
by seeding cofactors, thus allowing
-synuclein to aggregate at lower
concentrations than in our experiments. Furthermore, the formation of
fibrils may take years in vivo but happens within weeks in
our system. It will also be interesting to compare
-synuclein
fibrils found in Lewy bodies (6) with our synthetic
-synuclein
fibrils in the same experimental set up.
Most importantly, our results show that both point mutations causing
familial PD enhance the aggregation tendency observed in the wild type
protein. This is critical, because a pathogenesis model that can cover
all known mutants plus the wild type situation is less likely to be
based on non-disease-relevant effects. For example, in the Alzheimer's
disease field, the key evidence for the amyloid hypothesis is the
finding that all known early onset familial Alzheimer's disease
mutations have one common effect: they increase A
42 production
compared with the wild type (reviewed in Ref. 16). Our finding that all
forms of
-synuclein aggregate, but both mutants aggregate faster,
immediately suggests that the aggregation is relevant for the
pathogenesis of PD and may explain the early disease onset in these
families. Although the concentrations used here are nonphysiological,
the data show an effect that mirrors the biology in an assay that
accomplishes aggregation of all forms of
-synuclein, including wild
type. This is important, because most Lewy body diseases seem to
involve aggregation of wild type
-synuclein.
Our in vitro assay can be exploited for various
applications. The observation that the two mutants enhance aggregation
to different extents suggests that one could design additional
mutations that would enhance
-sheet formation and further accelerate
aggregation in vitro. As a first example, we tested the
A53T/A30P double mutant; in some experiments this analog aggregated
faster than each of the original mutants; however, this effect was not
always observed. Very rapidly aggregating mutant
-synuclein could be
critical in the generation of a transgenic model of Lewy body
formation. It will be important to see whether such mice develop
Parkinsonian symptoms. Finally, the system we have established here can
be readily adapted to high throughput screening for compounds that block
-synuclein aggregation. Such inhibitors could be useful as PD
therapeutics if aggregation of
-synuclein is a critical step in all
forms of PD.
 |
FOOTNOTES |
*
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.
These authors contributed equally to the work.
§
To whom correspondence should be addressed: Amgen, Inc., One Amgen
Center Dr., Thousand Oaks, CA 91320-1789. Tel.: 805-447-4520; Fax:
805-480-1347; E-mail: mcitron{at}amgen.com.
 |
ABBREVIATIONS |
The abbreviations used are:
PD, Parkinson's
disease;
FTIR, Fourier transform infrared spectroscopy.
 |
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