Molecular Biology of the Cell
Vol. 19, 1093–1103, March 2008
?-Synuclein–induced Aggregation of Cytoplasmic Vesicles
in Saccharomyces cerevisiae
James H. Soper,*†Subhojit Roy,*†Anna Stieber,*†Eliza Lee,*†Robert B. Wilson,†
John Q. Trojanowski,*†Christopher G. Burd,‡and Virginia M.-Y. Lee*†
*Center for Neurodegenerative Disease Research and Departments of†Pathology and Laboratory Medicine
and‡Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104
Submitted August 29, 2007; Revised December 12, 2007; Accepted December 20, 2007
Monitoring Editor: Jeffrey Brodsky
Aggregated ?-synuclein (?-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson’s disease (PD) and
related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies
have shown that when overexpressed in Saccharomyces cerevisiae, ?-syn localizes to plasma membranes and forms
cytoplasmic accumulations similar to human ?-syn inclusions. However, the exact nature, composition, temporal evolu-
tion, and underlying mechanisms of yeast ?-syn accumulations and their relevance to human synucleinopathies are
unknown. Here we provide ultrastructural evidence that ?-syn accumulations are not comprised of LB-like fibrils, but are
associated with clusters of vesicles. Live-cell imaging showed ?-syn initially localized to the plasma membrane and
subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of ?-syn
revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because
vesicular clustering is also found in LB-containing neurons of PD brains, ?-syn–mediated vesicular accumulation in yeast
represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies.
Parkinson’s disease (PD) is the most prevalent neurodegen-
erative movement disorder and is characterized by brady-
kinesia, rigidity, postural instability, and resting tremor
(Galvin et al., 2001; Forman et al., 2005), as well as by the loss
of dopaminergic neurons and presence of neuronal inclu-
sions, known as Lewy bodies (LBs) and Lewy neurites (For-
man et al., 2005). The identification of an ?-syn gene (SNCA)
mutation associated with autosomal dominant PD (Poly-
meropoulos et al., 1997), and the identification of fibrillar
?-syn as the principal component of LB pathology (Spillan-
tini et al., 1997), indicate that the LBs formed by ?-syn define
classic PD and are implicated in disease pathogenesis. The
subsequent identification of additional disease-linked SNCA
mutations (Polymeropoulos et al., 1997; Kruger et al., 1998;
Zarranz et al., 2004), as well as replications of the SNCA gene
(Singleton et al., 2003; Chartier-Harlin et al., 2004), indicate
that both mutant ?-syn and increased expression of wild-
type (WT) ?-syn can cause neurodegeneration.
In addition to PD, filamentous ?-syn inclusions have been
detected in other neurodegenerative diseases including the
LB variant of Alzheimer’s disease, dementia with LBs, neu-
rodegeneration with brain iron accumulation type-1, and
multiple system atrophy, which are collectively known as
synucleinopathies (Spillantini et al., 1997; Duda et al., 2000).
?-syn fibrils exhibit properties of amyloid (Spillantini et al.,
1998), and ?-syn assembles into amyloid fibrils in vitro (Han
et al., 1995; Giasson et al., 1999), whereas SNCA mutations
can accelerate ?-syn fibrillization (Conway et al., 1998;
Greenbaum et al., 2005). Therefore, it is plausible that ?-syn
misfolds and aggregates into amyloid fibrils that are neuro-
toxic and play a causative role in the pathogenesis of PD.
Thus, the elucidation of these processes could be exploited
for PD drug discovery.
?-syn, a short 140-amino acid protein (Iwai et al., 1995) is
localized to synaptic terminals (Maroteaux and Scheller,
1991) where it appears to play a role in regulating the distal
pool of presynaptic vesicles (Murphy et al., 2000, Cabin et al.,
2002) and dopamine release (Abeliovich et al., 2000) or func-
tions as a chaperone (Chandra et al., 2005). Furthermore, the
N-terminus of ?-syn contains several imperfect KTKEGV
repeats (Maroteaux et al., 1988; George et al., 1995; Weinreb
et al., 1996), which are thought to mediate binding to mem-
branes by interacting with phospholipids and shifting from
a random coil to an ?-helical conformation (Davidson et al.,
1998; Perrin et al., 2000; Kim et al., 2006). Interestingly, the PD
familial mutation A30P in ?-syn showed reduced binding to
membranes due to the Ala3Prosubstitution in repeat 2,
which disrupts membrane interaction (Jensen et al., 1998;
Cole et al., 2002). The central region of ?-syn is known as the
NAC domain, and it contains hydrophobic amino acids
required for fibrillization, particularly residues 71-82 (Gias-
son et al., 2001).
Over the past 10 years, multiple in vitro, animal, and cell
culture models have been developed to model LB formation
and elucidate mechanisms of LB-mediated neurodegenera-
tion. Recent studies have demonstrated that overexpressed
?-syn in Saccharomyces cerevisiae localizes to the plasma
membrane and forms inclusions (Outeiro and Lindquist,
This article was published online ahead of print in MBC in Press
on January 9, 2008.
Address correspondence to: Virginia M.-Y. Lee (vmylee@mail.
Abbreviations used: ?-syn, ?-synuclein; EM, electron microscopy;
ER, endoplasmic reticulum; GTPase, guanosine triphosphatase; PD,
Parkinson’s disease; WT, wild type.
© 2008 by The American Society for Cell Biology 1093
2003; Flower et al., 2005). Expression of ?-syn in yeast also
was shown to inhibit growth, induce accumulation of lipid
droplets, alter vesicle trafficking (Outeiro and Lindquist,
2003), increase reactive oxygen species (Flower et al., 2005),
and stimulate the heat shock response (Dixon et al., 2005).
Furthermore, proteins involved in endoplasmic reticulum
(ER)-to-Golgi trafficking may be involved in this ?-syn–
induced growth defect because this defect was rescued by
overexpressing Ypt1p, a Rab guanosine triphosphatase (GTPase;
Cooper et al., 2006). Interestingly, Ypt1p was also found to
associate with the ?-syn accumulations in yeast. To elucidate
this pathological process, we examined the temporal se-
quence of ?-syn accumulation in the cytoplasm of S. cerevi-
siae by following the changes in ?-syn-enhanced green fluo-
rescent protein (EGFP) localization using time-lapse microscopy.
We then identified distinct structural domains of ?-syn that
mediate this process. Importantly, ultrastructural studies
failed to detect ?-syn fibrils or LB-like inclusions but instead
identified the ?-syn accumulations as clusters of vesicles of
varying sizes. These vesicle clusters bear markers of secre-
tory vesicles as well as ER-Golgi transport vesicles, and
?-syn–induced vesicle accumulation also disrupted early
and late Golgi components. Because vesicles and membra-
nous profiles also accumulate at the periphery of LBs in PD
brain, ?-syn–induced vesicular clustering in the cytoplasm
of S. cerevisiae recapitulates features of PD that may underlie
mechanisms of neurodegeneration in PD.
MATERIALS AND METHODS
Yeast Strains and Media
The BY4742 (MAT ? his3?1 leu2?0 lys2?0 ura3?0) strain of S. cerevisiae was
used for all experiments in this study. Transformation of yeast was performed
using a standard lithium polyethylene glycol transformation procedure (Gietz
et al., 1992). Yeast cultures were grown in selective minimal media, containing
2% glucose or 2% galactose, deficient in the required amino acids. For induc-
tion of gene expression, cells were cultured in glucose media overnight, and
cells from this culture were taken to inoculate a second culture (starting
A600? 0.1) in galactose media, which was then grown at 30°C for 16 h.
Both 2? and integrative expression systems were generated and used for
experimentation. To generate full-length, truncated, or mutated ?-syn as well
as ?-syn plasmids, primer sequences for the corresponding cDNAs were
amplified from our mammalian pcDNA3.1 (Invitrogen, Carlsbad, CA) ?-syn
constructs. EGFP sequences were similarly amplified out of pIRES-EGFP
vector (Clontech, Palo Alto, CA). These ?-syn and EGFP products were
sequenced and digested with KpnI/HindIII and HindIII/XhoI, respectively,
and ligated into the MCS of the yeast vector, pYES2 (Invitrogen).
Integrative strains were generated by PCR amplification of the Gal1 pro-
moter, ?-syn protein sequence, transcription terminator, and selection marker
sequence out of the PYES2 vector. Primer sequences were chosen for directed
integration into the yeast genome 500 base pairs upstream of the Gal1 pro-
moter. For overexpression of 3HA-Ypt1p and 3HA-Sec4p, we used the
Growth was measured on solid media by making serial fivefold dilutions of
log-phase cultures, starting with A600? 0.4. Samples of each dilution were
spotted onto minimal media glucose and galactose plates, with appropriate
amino acids for selection. Plates were incubated at 30°C for 2–4 d.
Cell extracts were prepared as described previously (Kushnirov, 2000).
Briefly, cells were grown in galactose media to log-phase. Equal amounts of
cells were pelleted by centrifugation, resuspended in 1 volume water, and
treated with 1 volume 0.2 M NaOH for 10 min. Cells were pelleted again,
resuspended in SDS-PAGE buffer, and boiled for 5 min, and protein samples
were separated by SDS-PAGE.
For immunoblotting, we used LB509, an anti-?-syn mouse mAb recognizing
residues 115-122 (Jakes et al., 1999), Syn303, an anti-?-syn mAb that recognize
an epitope containing residues 1-5 of ?-syn (Duda et al., 2002); MAB2510, a
GFP mAb (Chemicon, Temecula, CA); Syn 207, an anti-?-syn mAb (Giasson et
al., 2000); MAB1864, an anti-?-tubulin antibody (Chemicon); and Ab4645, an
anti-Pma1 antibody (Abcam, Cambridge, MA).
Membrane fractionation was performed as previously described (Juschke et
al., 2004). Briefly, logarithmically growing cells were washed in 20 mM
HEPES, pH 7.5, 20 mM NaF, and 20 mM NaN3. Cells were lysed in 25 mM
Tris/HCl, pH 8.0, 2.5 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride,
with protease inhibitors, by vortexing with glass beads. Unbroken cells were
cleared by centrifugation at 1200 ? g for 2 min. The lysate was then subjected
to centrifugation at 25,000 ? g for 20 min. The supernatant was retained as the
cytosolic fraction, and the pellet was resuspended in 0.55 ml of 10 mM
Tris/HCl, pH 7.4, 0.2 mM EDTA, 0.2 mM dithiothreitol, 20% glycerol, and 0.5
ml was loaded on top of a sucrose gradient (0.5 ml 53% sucrose, 1 ml 43%
sucrose), and centrifuged for 2 h at 100,000 ? g. Six 320-?L fractions were
taken from the top, precipitated with an equal volume 20% trichloroacetic
acid, and analyzed by SDS-PAGE gels and immunoblotting.
Live Imaging and Accumulation Quantification
Yeast cultures were grown to midlog phase in minimal media containing
glucose, with the appropriate amino acids for selection. Cells were then
washed once with water and once in galactose minimal media. Cells were
then resuspended in galactose minimal media and plated onto PDL-coated
glass-bottom dishes (MatTek, Ashland, MA). The minimal media was supple-
mented with Oxyrase (1:100, Oxyrase, Mansfield, OH) to minimize photo-
damage during fluorescence imaging. Warmed Oxyrase-containing medium,
1.5 ml, was added to the dish, and the lid was sealed with parafilm. The dish
was then transferred on to a custom-built Plexiglas incubator housed over a
Nikon TE-2000-E (Nikon, Tokyo, Japan) inverted epifluorescence microscope.
The incubator was kept at ?30°C using an Air-Therm ATX temperature
controller (World Precision Instruments, Sarasota, FL). To further minimize
photodamage and photobleaching, the intensity of the fluorescence lamp was
reduced to ?15% of total intensity using neutral-density filters. Under these
conditions, yeast displayed normal growth and budding for the entire imag-
ing session (10–16 h). Typically the exposure time was between 100 and 2000
ms, with the camera (CoolSnap-HQ, Photometrics, Tucson, AZ) operating at
maximum gain. Images were typically captured at 60? or 100? magnifica-
tion. All image acquisition and processing was done using Metamorph (Mo-
lecular Devices, Downingtown, PA). All images were scaled linearly in Meta-
morph and subsequently mounted in Adobe Photoshop (Adobe, San Jose,
CA) for display.
Quantification of accumulations was done by capturing images of cultures
that had been grown in galactose media for 16 h. Four fields from separate
cultures were counted, with ?250 cells in each field, and cells were scored for
presence of accumulations.
Fixation was done by mixing equal volumes of cold 2? fixative and yeast cell
suspensions, fixing for 5 min on ice, pelleting and resuspending in cold 1?
fixative, and continuing fixation on ice for a total of 35 min. The final (1?)
concentration of the fixative was 2% glutaraldehyde in 40 mM KPO4buffer,
pH 6.7, 1 mM MgCl2,1 mM EGTA, and 1.0 M sorbitol. Cells were washed in
decreasing concentrations of sorbitol in 40 mM KPO4buffer, washed 10 min
in 50 mM NH4Cl, and stored overnight in buffer. The next day, cells were
treated for 10 min with freshly made 1% Na-m-periodate, rinsed in DH2O,
and washed for 10 min in 50 mM NH4Cl. They were postfixed for 20–30 min
in 0.5% OsO4? 0.8% K ferrocyanide in 0.1 M cacodylate buffer, pH 7.4, on ice,
dehydrated in cold ethanol, embedded in LR White resin, and polymerized
for 2 d at 45–47°C. Thin sections were stained for 10–20 min with bismuth
For immunostaining, thin sections were picked up on formvar-coated grids
and immunostained floating on drops of solution. The buffer used was 10 mM
tris-HCl in PBS ? 0.02% NaH3.The primary antibodies used were SNL1, a
rabbit polyclonal antibody to ?-syn and normal rabbit IgG. Antibody dilu-
tions and blocking solution were spun for 5 min at 13,000 rpm on a microfuge
before use. Sections were blocked for 45 min in 1% ovalbumin ? 0.2% fish
gelatin in buffer (block), incubated overnight at 4C in primary antibody in 1:10
dilution of block, washed five times for 5 min on buffer, blocked for 5 min,
and incubated for 2.5–4 h on anti-rabbit IgG coupled to 10-nm colloidal gold
in 1:10 block. They were washed three times with buffer, three times with
dH2O, dried, and stained for 10–20 min with bismuth subnitrate.
Electron microscopy was also conducted on samples of substantia nigra
from a patient with PD that was harvested 2 h postmortem, fixed overnight
in 4% paraformaldehyde and 0.1% glutaraldehyde, and cut into 50-?m sec-
tions with a Vibratome. The sections were fixed for an additional 10 min in 2%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, postfixed for 30 min in 1%
OsO4? 1.5% potassium ferrocyanide in buffer, dehydrated, and embedded in
J. H. Soper et al.
Molecular Biology of the Cell 1094
Cells were grown in galactose minimal media for 16 h and fixed in 4%
paraformaldehyde in 40 mM phosphate buffer for 1 h at 30°C. Cells were then
washed twice in 40 mM phosphate buffer containing 1.2 M sorbitol (sorbitol
buffer) and incubated for 10 min at 30°C in sorbitol buffer containing 300
?g/ml zymolyase 100T (Seikagaku America, Rockville, MD) to digest the cell
wall. Cells were then washed twice in sorbitol buffer and once in PBS
containing 1% BSA, 0.5% Tween (Block buffer). Cells were then incubated
with block buffer containing the appropriate dilution of primary antibody at
4°C overnight, washed twice in block buffer and incubated with secondary
antibody for 1 h at room temperature. Cells were finally washed three times
in block buffer and mounted on PDL-coated dishes for microscopy. Antibod-
ies used for immunofluorescence were LB509, Syn303, and 12CA5, an anti-
hemagglutinin (HA) antibody (Roche, Indianapolis, IN).
Genomic Red Fluorescent Protein Tagging
Endogenous proteins were red fluorescent protein (RFP) tagged as described
(Sheff and Thorn, 2004). Briefly, sequence-specific primers were used to
amplify mCherry RFP or dTomato dimeric RFP with the HISMX selection
cassette and site-specific sequences for directed integration. These PCR prod-
ucts were used directly to transform yeast cells, which were then grown in
selective media, and screened by fluorescence microscopy to confirm integra-
tion of the RFP tags.
Quantification of Sec7p and Cop1p disruption was measured in a blinded
experiment by examining 10 fields from three different samples with ?100
cells per field. Sec7p disruption was first measured by examining the red
channel. Cells with one or more discrete Sec7 punctate structures were
considered normal, and cells with 0-labeled structures were considered dis-
rupted. Cells containing large ?-syn-EGFP accumulations were identified by
examining the green channel. Cells with inclusions greater than or equal to
10% of the cell diameter were considered cells bearing large accumulations.
These cells were then matched to the corresponding red channel image to
examine Sec7p disruption.
Comparison of sample means and calculation of P values was performed
using GraphPad software (San Diego, CA).
?-Synuclein Accumulation in the Cytoplasm of S.
cerevisiae Is Initiated at the Plasma Membrane
To gain insight into the sequence of events that lead to ?-syn
accumulation in the cytoplasm of S. cerevisiae, yeast strains
expressing human WT and familial PD ?-syn mutants with
a C-terminal EGFP tag under control of a galactose-induc-
ible, glucose-repressible promoter (?-syn-EGFP, A53T-EGFP
and A30P-EGFP, respectively) were first examined in the 2?
pYES2 expression system and subsequently were integrated
into the yeast genome upstream of the Gal1 promoter. Both
integrated and nonintegrated ?-syn constructs yielded sim-
ilar results. Furthermore, a number of artificial ?-syn mu-
tants (Figure 1A) with N- or C-terminal truncations (denoted
as 58-140-EGFP and 1-57-EGFP, respectively) as well as the
fibrillization incompetent ?-syn mutant, ?71-82-EGFP (Gi-
asson et al., 2001) were also generated. The latter mutants
were used in the structure-function studies described below.
To monitor expression of the various ?-syn-EGFP con-
structs, immunoblot analysis was conducted using an anti-
C-terminal–specific ?-syn antibody (LB509), an anti-N-ter-
minal antibody (Syn303), and an anti-EGFP antibody. All
antibodies detected a major protein band with MW ?50 kDa
and some minor breakdown products, suggesting the entire
?-syn-EGFP fusion protein is expressed in the WT and mu-
tant ?-syn-EGFP cells (Figure 1B). An ?-syn S129 phospho-
specific antibody detected a phosphorylated isoform of
?-syn-EGFP from cells expressing WT syn-EGFP, A53T-
EGFP, A30P-EGFP, ?71-82-EGFP, and 58-140-?-syn-EGFP
(data not shown). ?-Syn phosphorylation at S129 in yeast is
unusual possibly because this site is not highly phosphory-
lated under physiological conditions (Okochi et al., 2000),
but it is heavily phosphorylated in LBs of PD and other
synucleinopathies (Fujiwara et al., 2002).
We also performed immunofluorescence using our anti-C
(LB509) and anti-N (Syn303) terminal specific ?-syn anti-
bodies (Figure 1C) and showed that both antibodies com-
pletely colocalized with the EGFP fluorescence. As previ-
ously reported (Outeiro and Lindquist, 2003), untagged
?-syn localized similar to EGFP-tagged ?-syn in yeast
(data not shown).
As shown previously (Outeiro and Lindquist, 2003;
Zabrocki et al., 2005; Dixon et al., 2005), ?-syn-EGFP promi-
nently localized to plasma membranes and formed cytoplas-
mic accumulations, whereas EGFP alone showed a diffuse
cytoplasmic distribution (Figures 1A and 2A). The ?-syn
clusters/accumulations varied considerably in size, and
many of the smaller ones appeared to be associated with the
cell cortex, whereas larger accumulations were sometimes
completely localized to the cytoplasm, suggesting that they
may form initially at the plasma membrane or the ER, which
underlies the plasma membrane in yeast (Figure 1D). The
binding of ?-syn-GFP to plasma membrane was further
confirmed biochemically in membrane fractionation studies.
Although both ?-syn-EGFP and EGFP were detected in the
cytosolic fraction, a significant portion of ?-syn-EGFP but
not EGFP was recovered in the plasma membrane fractions
(Supplementary Figure 1).
To determine the temporal sequence of the cytoplasmic
accumulation of ?-syn-EGFP, we performed live imaging
studies on yeast cells expressing WT ?-syn-EGFP. After
induction with galactose, initial expression of the protein
was observed at the cell cortex (Figure 1D). This was fol-
lowed by the accumulation of small ?-syn-EGFP aggregates
at the cell cortex ?80 min later. Time-lapse microscopy
demonstrated that the small accumulations grew in size and
sometimes merged together to form larger accumulations
that often appeared to detach from the cell cortex and as-
semble into cytoplasmic clusters (Figure 1D and Supplemen-
tary Information, Video 1 online). These experiments pro-
vide direct evidence indicating that membrane localization
may be a required first step in the formation of ?-syn accu-
mulations in yeast cells.
?-Synuclein Accumulation in the Cytoplasm Requires
Both the N-terminus and the NAC domain
Previous studies have shown that the N-terminal KTKEGV
imperfect ?-helical motifs are involved in membrane bind-
ing (Davidson et al., 1998; Perrin et al., 2000; Kim et al., 2006),
but it is unclear if they are required for ?-syn localization to
the plasma membrane. To test this possibility, we generated
yeast cell lines expressing ?-syn-EGFP fusion proteins with
either the first 57 amino acids alone (1-57-EGFP) or with
these residues deleted (58-140-EGFP; Figures 1A and 2A).
Significantly, ?-syn constructs with the N-terminus alone
(1-57-EGFP) localized to the plasma membrane (Figure 2A),
whereas ?-syn constructs with an N-terminus deletion (58-
140-EGFP) showed a diffuse cytoplasmic distribution with
no membrane localization (Figures 2A and Supplementary
Information, Video 2 online). A53T-EGFP, ?71-82-EGFP,
and ?-syn-EGFP all localized to the membrane, consistent
with the fact that these all contain the N-terminal ?-helical
repeats. Although A30P-EGFP was localized to the mem-
brane, it did not form accumulations in the cytoplasm (Fig-
ures 2A and Supplementary Information, Video 3 online);
this may be due to the substitution of alanine by a proline, a
helix breaker, at the end of repeat 2. Consistent with this
hypothesis, we observed substantial diffuse cytoplasmic
A30P-EGFP, suggesting that this mutation may decrease
membrane binding. However, an alternative explanation
could be that A30P ?-syn is specifically targeted to the
?-Synuclein-induced Vesicular Accumulation
Vol. 19, March 2008 1095
vacuole for degradation by the yeast protein YPP1 (Flowers
et al., 2007). Thus, our data demonstrated the requirement
for the ?-syn N-terminus and most likely the helical repeats
therein for membrane localization.
The central hydrophobic NAC region of ?-syn has been
implicated as a crucial region for amyloid fibril formation
and ?-syn self-assembly (El Agnaf et al., 1998; Giasson et al.,
2001). We determined if this region is also important for the
formation of ?-syn accumulations in this yeast model by
generating yeast lines expressing a ?-syn-EGFP fusion pro-
tein lacking amino acids 71-82 of ?-syn (?71-82-EGFP, Fig-
ures 1A and 2A). Although ?71-82-EGFP localized to the
membrane at levels comparable to WT ?-syn-EGFP, we
observed a 10-fold decrease in the number of cells that
developed ?-syn accumulations (i.e., 2–3% in ?71-82 vs.
30–35% in syn-EGFP cells, Figures 2B and Supplementary
Information, Video 4 online). Furthermore, A53T-EGFP
(which contains the entire NAC domain) but not ?-syn-
EGFP (which lacks residues 71-82) formed cytoplasmic ac-
cumulations. These results support an important role for
residues 71-82 in mediating ?-syn accumulation.
To evaluate whether or not membrane binding was suffi-
cient to cause ?-syn-EGFP accumulations, we compared the
distribution of EGFP with the different syn-tagged EGFP
fusion proteins 16 h after galactose induction. As expected,
1-57-EGFP was localized to the membrane, but there were
of KTKEGV motif repeats, the hydrophobic residues 71-82 within the NAC domain, A30P and A53T point mutations, and truncated/deleted
regions are indicated. (B) Immunoblot analysis of yeast extracts from cells expressing PYES2 constructs using a C-terminal anti-?-syn mAb
(LB509), an N-terminal anti-?-syn mAb (Syn303), an anti-GFP antibody, an anti-?-syn mAb (Syn 207), and an ?-tubulin antibody. (C) ?-Syn
immunofluorescence on yeast expressing ?-syn-EGFP using LB509 and Syn303. Both mAb immunostaining colocalize completely with the
GFP signal, indicating that the entire fusion protein is present. (D) Time-lapse imaging of ?-syn-EGFP (PYES2 Syn-EGFP) accumulation
formation in yeast. Images were taken every 80 min starting when syn-EGFP protein expression was visible. Membrane-associated
accumulations begin to form at 80 min (arrow) and grow larger in size. Some accumulations appear to coalesce to form larger accumulations
(arrowheads). For corresponding video, see Supplementary Information, Video1 online. Scale bars, (C) 2 ?m; (D) 1 ?m.
?-Syn-EGFP expression and accumulation in S. cerevisiae. (A) Schematic of ?-syn-EGFP constructs used in this study. Locations
J. H. Soper et al.
Molecular Biology of the Cell1096
no accumulations (Figure 2A), suggesting that membrane
binding alone is not sufficient to cause formation of accu-
mulations. Similarly, although ?71-82-EGFP was distributed
to the membrane, only a small number of cytoplasmic ?-syn
clusters were detected. The N-terminal deletion, 58-140-
EGFP, was not localized to the membrane and ?-syn accu-
mulations were not found. Thus, these data suggest that
membrane binding is a necessary step for the formation of
cytoplasmic accumulations of ?-syn in yeast, but this step
alone is not sufficient for accumulation to occur. Taken to-
gether, our time-lapse microscopy and structure-function
experiments provide evidence that the accumulation of cy-
toplasmic ?-syn occurs sequentially through a two-step pro-
cess: The first step involves membrane binding, which prob-
?-syn, and the second step requires the hydrophobic NAC
domain to drive accumulation of ?-syn-EGFP.
Finally, to evaluate if the presence of ?-syn accumulations
results in toxicity (Outeiro and Lindquist, 2003, Cooper et al.,
2006), we performed growth assays to examine the effect of
?-syn domain deletions on cell growth. As previously re-
ported, ?-syn-EGFP had a mild toxic effect on cell growth,
and this effect was not observed with any of the ?-syn
deletion mutants or with ?-syn, all of which did not form
accumulations (Figure 2C). Our results support a role for
?-syn accumulation in the inhibition of cell growth, because
the elimination of these accumulations, either through elim-
ination of membrane binding or deletion of the NAC region,
abolishes this toxicity.
?-Syn-EGFP Accumulations Are Composed of Clusters of
Vesicles in the Cytoplasm
To determine if ?-syn-EGFP accumulations resemble PD-
like LBs, we performed electron microscopy (EM) on the
?-syn-EGFP accumulations. To facilitate imaging of these
?-syn accumulations, we utilized cells with ?-syn-EGFP in-
tegrated into the yeast genome. These cells produced twice
the percentage of cytoplasmic ?-syn accumulations as the
nonintegrated strain (Supplementary Figure 2). The number
of cells with ?-syn accumulations was further enhanced to
?70% by treatment of these yeast with 5% DMSO (Zabrocki
et al., 2005). Surprisingly, EM analyses of the cells expressing
?-syn-EGFP did not reveal filamentous ?-syn- or LB-like
structures. Instead, large accumulations of membranous
vesicles, ranging in size from 20 to 100 nm in diameter were
observed (Figure 3, A–C). These vesicular clusters were
observed in cells expressing ?-syn-EGFP and A53T-EGFP,
but were not detected in cells expressing 1-57-EGFP, 58-140-
EGFP, or EGFP alone, thereby indicating that these vesicular
clusters are integral components of the ?-syn accumulations
visualized by fluorescence microscopy (Figure 3, compare
panels A–C and G with panels D–F and H). Similar vesicular
accumulations were also observed in cells grown without
DMSO (Figure 3I), and in cells expressing untagged ?-syn
(Supplementary Figure 3).
To confirm that ?-syn is indeed present within the vesic-
ular clusters, immuno-EM was conducted using a polyclonal
anti-?-syn antibody (SNL-1). Immunogold particles (local-
ized ?-syn) on or near vesicles within a cluster in ?-syn-
EGFP–expressing cells suggesting that ?-syn is bound to the
vesicles (Figure 3, J–L). As expected, plasma membrane
localization of ?-syn-EGFP immunogold particles was also
evident (Figure 3, J–L). However, no plasma membrane
localization or vesicular accumulations were detected in
cells expressing 58-140-EGFP (Figure 3H). Elimination of the
osmication resulted in more robust immunolabeling of
?-syn in the plasma membrane and accumulations, but
poorer preservation of organelle and vesicle structures (Fig-
Our ultrastructural studies demonstrate that these ?-syn-
EGFP clusters are a collection of vesicular structures con-
taining ?-syn and are not ?-syn amyloid fibrils that are the
defining features of LBs in PD brains. This striking observa-
tion suggests a new mechanism for ?-synuclein cellular
toxicity that may be relevant to PD.
accumulation and toxicity in yeast. (A) Effect of de-
letions and mutations on ?-syn-EGFP localization in
yeast. EGFP alone has a diffuse localization, whereas
WT ?-syn-EGFP binds the plasma membrane and
forms membrane associated accumulations. Dele-
tion of the N-terminus (58-140-EGFP) eliminates
membrane binding and ?-syn accumulation, whereas
expression of only the N-terminus (1-57-EGFP) pre-
serves membrane binding but eliminates ?-syn ac-
cumulation. The deletion of residues 71-82 (?71-82-
EGFP), a region important for ?-syn fibril formation,
showed a dramatic reduction in the number of cells
with ?-syn accumulation although membrane local-
ization is not impaired. Similarly, ?-syn-EGFP also
binds membrane but does not accumulate in the
cytoplasm. A30P-EGFP shows membrane localiza-
tion and cytoplasmic distribution but does not accu-
mulate. (B) Quantification of the percentage of cells
containing accumulations in yeast expressing differ-
ent deletions and mutations of ?-syn-EGFP. Error
bars, SD. (C) Effect of various deletions on growth of
yeast. Only cells expressing full-length ?-syn have a
growth defect. Scale bar, (A) 1 ?m.
Structural requirements for ?-syn-EGFP
?-Synuclein-induced Vesicular Accumulation
Vol. 19, March 2008 1097
?-Syn Vesicular Clusters Contain Both Secretory Vesicles
and ER-Golgi Transport Vesicles
To provide insights into the identity of the vesicles detected
by EM, we evaluated the localization of HA-tagged Sec4p, a
Rab GTPase which localizes to secretory vesicles (Guo et al.,
1999) and HA-tagged Ypt1p, a Rab GTPase involved in
ER-Golgi transport that normally localizes to ER-Golgi
transport vesicles (Morsomme and Riezman, 2002) with
?-syn-EGFP. HA-tagged Sec4p colocalized with vesicular
accumulations in cells expressing ?-syn-EGFP (Figure 4A),
whereas it had a diffuse granular localization in cells ex-
pressing EGFP alone. Similar to a recent study (Cooper et al.,
2006), ?-syn-EGFP accumulations in yeast cells also colocal-
ize with Ypt1p (Figure 4B). To demonstrate the specificities
of HA-Sec4p and HA-Ypt1p in localizing to the correct
vesicles, we examined the distribution of HA-tagged Vps29p, a
peripheral membrane protein involved in endosome-to-
Golgi retrograde transport (Seaman et al., 1998), in EGFP and
?-syn-EGFP expressing cells. Significantly, we found no dif-
ference in Vps29p localization (Figure 4C), indicating that
the colocalization seen with HA-Sec4p and HA-Ypt1p is not
an artifact of the HA tag itself. Thus, our experiments sug-
gest that the ?-syn accumulations are comprised of vesicles
of multiple types, some of which contain secretory vesicle
and ER-Golgi transport vesicle markers.
Vesicular Clustering Caused by ?-Syn-EGFP Expression
Disrupts Organization of the Golgi
To determine if the presence of large vesicular accumula-
tions in cells expressing ?-syn-EGFP disrupt the localiza-
tion and function of other organelles, we examined the
organization of the ER, Golgi, vacuole, and endosomes by
genomically integrated RFP-tagged organelle markers in
cells containing ?-syn-EGFP vesicular accumulations. Us-
ing an RFP-tagged Sec63p, a protein involved in the trans-
location of polypeptides into the ER (Feldheim et al., 1992),
as a marker for yeast ER, we examined the localization of the
ER in yeast cells expressing EGFP and ?-syn-EGFP. A sim-
ilar discontinuous distribution of Sec63p around the plasma
membrane and also around the nucleus (Deshaies and
Schekman, 1990) was observed in control and ?-syn–ex-
pressing cells (Figure 5A), suggesting that the ER is not
disrupted in cells with vesicular accumulations. The local-
ization of the vacuole was also not disrupted by ?-syn-EGFP
accumulations, as shown in yeast cells expressing an RFP-
tagged Vma4p (Figure 5B), a subunit of the vacuolar ATPase
pump (Foury, 1990). The integrity of the endosomes is
posed of vesicles. Electron microscopy on yeast ex-
pressing ?-syn-EGFP shows dense accumulations of
vesicles (A–C), which do not appear in cells express-
ing EGFP alone (D). Cells expressing ?71-82-EGFP
(E), 1-57-EGFP (F), and 58-140-EGFP (H) do not have
vesicular accumulations. Cells expressing A53T-EGFP
have vesicular accumulations that are similar to wild
type (G). Cells expressing ?-synuclein without DMSO
treatment formed similar accumulations (I). Im-
muno-EM using SNL-1, a polyclonal anti-?-syn anti-
body with 10-nm colloidal gold, shows that ?-syn lo-
calizes to the vesicular accumulations (J–L), as well as
the plasma membrane (J and L). Omission of osmica-
tion resulted in stronger antibody staining (L). Cells
expressing EGFP alone had no staining with the anti-
?-syn antibody (D), whereas cells expressing 58-140-
EGFP displayed cytoplasmic staining (I). N, nucleus.
Scale bars, (A, D–I, and L) 500 nm; (B and J) 100 nm; (C
and K) 50 nm.
?-Syn-EGFP accumulations are com-
J. H. Soper et al.
Molecular Biology of the Cell1098
also unaffected, because ?-syn-EGFP expression in a
strain of yeast with dimeric-RFP-tagged Vps17p (Figure
5C), a membrane protein involved in endosome-to-Golgi
transport (Seaman et al., 1998), did not cause any change
in Vps17p distribution when compared with cells express-
ing EGFP alone.
To determine if ?-syn-EGFP vesicular accumulations af-
fect the organization of the Golgi, we examined the localiza-
tion of RFP-tagged Sec7p, a guanine nucleotide exchange
factor that localizes to late Golgi (Sata et al., 1998). In cells
expressing EGFP alone, Sec7p was localized to small punc-
tate structures around the periphery of the cell. However, in
cells containing ?-syn-EGFP vesicular accumulations, the
with Rab GTPases Sec4p and Ypt1p. (A) ?-Syn-EGFP accumulations
are colocalized with Sec4p. 3HA-tagged-Sec4p was overexpressed
(pRS413 3HA-Sec4p) in cells expressing either EGFP or ?-syn-EGFP.
In cells expressing EGFP or in ?-syn-EGFP cells without accumula-
tions, Sec4p was localized to small punctate structures throughout
the cytoplasm (secretory vesicles). In cells with ?-syn-EGFP accu-
mulations, Sec4p staining was strongly colocalized with these accu-
mulations. (B) ?-Syn-EGFP accumulations are colocalized with
Ypt1p. 3HA-tagged-Ypt1p was overexpressed (pRS413 3HA-Ypt1p)
in cells expressing either EGFP or syn-EGFP. Cells were immuno-
stained with anti-HA antibody. In cells expressing EGFP or ?-syn-
EGFP cells without accumulations, Ypt1p is localized to small punc-
tate structures (most likely ER-Golgi transport vesicles) throughout
the cytoplasm, whereas in cells with ?-syn-EGFP accumulations,
Ypt1p colocalizes with these accumulations. (C) 3HA-tagged-
Vps29p did not colocalize with ?-syn-EGFP accumulations, and
localization of Vps29p was similar in cells expressing EGFP and
?-syn-EGFP. Scale bar, 2 ?m.
?-Syn-EGFP vesicular accumulations are colocalized
the ER, vacuole, or endosomes. (A) Presence of ?-syn-EGFP accumu-
lations does not disrupt localization of the ER marker Sec63p. Sec63p
was C-terminally tagged with monomeric RFP. Expression of EGFP or
?-syn-EGFP shows similar localization of the ER marker. (B) Presence
of ?-syn-EGFP accumulations does not disrupt localization of the
vacuole membrane marker Vma4p, which was c-terminally tagged
with monomeric RFP. (C) Presence of ?-syn-EGFP accumulations does
not disrupt localization of the endosomal marker, Vps17p, which was
c-terminally tagged with dimeric RFP. Scale bar, 2 ?m.
?-Syn-EGFP accumulations do not disrupt organization of
?-Synuclein-induced Vesicular Accumulation
Vol. 19, March 20081099
Sec7p localization was disrupted, displaying a complete lack
of discrete Sec7p puncta (Figure 6A). We quantified the
disruption of Sec7p by comparing the localization of Sec7p
in cells containing large (greater than or equal to 10% of the
cell diameter) ?-syn-EGFP accumulations with cells express-
ing EGFP alone. We found that there was a substantial increase
(p ? 0.001) in Sec7p disruption from cells expressing EGFP
alone (14.8 ? 5.7%) to cells containing large ?-syn-EGFP
accumulations (60.1 ? 15.1%; Figure 6B). Cells expressing
?-syn-EGFP without large accumulations displayed normal
Sec7p localization, indicating that the formation of accumula-
tions and not simple expression of ?-syn is required for Sec7p
disruption. A second Golgi marker, Cop1p, was examined and
found to be disrupted in cells expressing Syn-EGFP compared
with controls (Supplementary Figure 4, 67.2 ? 1.4% increase
from control, p ? 0.0001).
Taken together, these data show that ?-syn vesicular ac-
cumulations in yeast results in a profound disruption of
Golgi morphology, as indicated by Sec7p and Cop1p local-
ization, but does not affect the localization of markers of ER,
vacuole, or endosomes.
Evidence of Vesicular Accumulations in Human LBs
Our experiments thus far demonstrate that ?-syn expression
in yeast resulted in cytoplasmic accumulations of vesicles
that are associated with toxicity. To determine if similar
vesicular accumulations are found in LBs of PD, we exam-
ined the ultrastructure of LBs from the substantia nigra
(from a 66-y-old PD patient) with a short postmortem inter-
val and that was optimally fixed for EM. In this case, many
typical LBs were observed (Figure 7, A–C). Additionally, we
observed dense clusters of small vesicles around the perim-
eter of these LBs (Figure 7, A–E). These vesicles ranged in
size between 20 and 100 nm. They were typically found
around the edges of the LBs, away from the densely packed
fibrils (Figure 7E). Examination of an area more distal to the
LB revealed a lack of dense clusters of vesicles, although
sparse vesicles that appear to be of similar size are visible
(Figure 7F). These data are consistent with the earlier EM
studies of LBs (Duffy and Tennyson, 1965) and suggest that
vesicle accumulations, similar to those we report here in our
yeast model system, may be important in the initial forma-
tion of LBs and the pathogenesis of PD.
Here, we have examined the process of ?-syn-EGFP accu-
mulation in yeast. We have demonstrated that ?-syn accu-
mulation requires an intact N-terminus as well as the NAC
domain and that clustered vesicles are integral components
of ?-syn accumulations similar to those observed in authen-
tic LBs in PD. The consequences of these ?-syn accumula-
tions in yeast include the disruption of the Golgi, as well as
the disruption of ER-to-Golgi and secretory vesicular traf-
ficking, and cellular toxicity. Importantly, the overexpres-
tion of the Golgi marker Sec7p. (A) Presence of ?-syn-EGFP accumu-
lations disrupt localization of the late Golgi marker Sec7p, which was
C-terminally tagged with monomeric RFP. Scale bar, 2 ?m (B) Quan-
tification of Sec7p disruption in cells expressing EGFP and cells ex-
pressing ?-syn-EGFP with large accumulations. Scale bar, 2 ?m.
Presence of ?-syn-EGFP accumulations disrupts localiza-
EM micrographs of LBs from the substantia nigra of a PD patient.
LBs were composed of a dense fibrillar core with associated small
vesicular accumulations (arrow) around the perimeter (A–E). Ex-
amination of sites more distal to the LBs revealed less dense distri-
bution of small vesicles than that seen around the LB perimeter (F).
Scale bars, (A, C, and E) 2 ?m. (B, D, and F) 500 nm.
Presence of vesicular accumulations in human PD LBs.
J. H. Soper et al.
Molecular Biology of the Cell1100
sion of ?-syn in yeast cells did not produce LB-like filamen-
of vesicles as in PD LBs. Although not a direct model for
recapitulating insoluble inclusions containing amyloid
?-syn, this model may provide insights into the potential
function of ?-syn and/or the early steps in the formation of
LBs because similar vesicular accumulations are detected in
human LBs of PD.
On induction with galactose, ?-Syn-EGFP was first local-
ized to the cell cortex. This interaction with membranes
appears to be dependent upon the N-terminal ?-helical re-
peats of ?-syn, because deletion of this subdomain abrogates
this early membrane localization. This is consistent with
previous studies demonstrating that these N-terminal re-
peats bind phospholipids and lipid membranes and may be
critical for the normal function of ?-syn (Perrin et al., 2000;
Kim et al., 2006) and that mutations in this region perturbs
plasma membrane localization in yeast (Volles and Lans-
bury, 2007). Hence, we propose that the binding of ?-syn to
the yeast plasma membrane may mimic its ability to bind
synaptic vesicles in neurons and may be a useful system to
study the membrane-binding function of ?-syn. Further-
more, we show here that membrane localization of ?-syn is
required for the subsequent recruitment of vesicles, because
the deletion of N-terminal repeats eliminated this membrane
localization as well as vesicular clustering.
However, our data also support the view that membrane
localization alone is insufficient to cause vesicle clustering
because expressing the first 57 amino acids of ?-syn is suf-
ficient to cause membrane localization, but not vesicle accu-
mulation. We also determined that deletion of the hydro-
phobic residues 71-82 within the NAC domain greatly
reduced the ability of ?-syn to induce vesicular clustering in
yeast, without affecting membrane localization. Thus, the
formation of vesicular accumulations may depend on the
ability of ?-syn to form intermolecular interactions through
the hydrophobic NAC domain. Alternatively, this region
may be important for ?-syn to assume a conformation
change that facilitates vesicle interaction.
Our EM studies revealed that ?-syn-EGFP accumulations
are composed of collections of vesicles. These vesicular clus-
ters do not contain fibrils, unlike authentic human LBs.
However, alterations in synaptic vesicle numbers due to
manipulation of ?-syn levels have been reported, including
evidence that suppression of ?-syn expression in cultured
neurons reduced of the distal pool of synaptic vesicles (Mur-
phy et al., 2000), that ?-syn knockout mice demonstrate an
impairment in the maintenance of the reserve pool of syn-
aptic vesicles (Cabin et al., 2002), and that overexpression of
?-syn in PC12 cells results in an accumulation of “docked”
vesicles at the synapse (Larsen et al., 2006). ?-Syn–induced
accumulation of vesicles in yeast may mimic the ability of
?-syn to regulate synaptic vesicles in neurons. Therefore, S.
cerevisiae may be a useful system for studying the role of
?-syn in the regulation and maintenance of synaptic vesicle
The presence of ?-syn–induced vesicular accumulations
caused a severe disruption of the organization of the Golgi
apparatus, but did not disrupt the ER, vacuole, or endo-
somes. A previous study showed that expression of ?-syn
caused disruption of ER-Golgi transport and cytotoxicity
(Cooper et al., 2006), whereas our data suggest that disrup-
tion of the Golgi organization may contribute to this pheno-
type. Golgi fragmentation has been observed in nigral neu-
rons with ?-syn–positive LBs, particularly in neurons containing
pale bodies that are thought to represent an early stage of
LBs formation (Fujita et al., 2006), suggesting that the Golgi
fragmentation caused by ?-syn expression in yeast may be
relevant to abnormalities in the human disease. Golgi frag-
mentation has also been observed as a consequence of pre-
fibrillar ?-syn aggregates in cell culture (Gosavi et al., 2002).
These two observations, in addition to our data, suggest that
?-syn could contribute to toxicity even when it is not in a
fibrillar state and that these vesicular accumulations may
represent early stages of LB formation as well as PD patho-
genesis. Thus, the formation of similar vesicular accumula-
tions in neurons could serve as a concentration point or
scaffold for the formation of ?-syn filaments and subsequent
One interesting observation in this yeast system is that the
familial ?-syn mutations, A30P and A53T, do not enhance
vesicular accumulation. In fact, A30P ?-syn does not form
vesicular accumulations in yeast. Currently, it is unclear
how A30P causes familial PD. The impaired binding to rat
brain vesicles (Jensen et al., 1998) and in yeast cells suggest
that it could lead to a reduction in ?-syn transport to pre-
synaptic terminals and a loss of function in the asymmetric
neuron. On the other hand, A53T ?-syn was shown to en-
hance fibrillization in both test tube studies and in trans-
genic mouse models of synucleionopathies when compared
with WT ?-syn (Conway et al., 1998, Giasson et al., 1999,
2002). Thus, the lack of increased vesicle clustering in yeast
cells expressing the A53T mutant suggests that this mutation
does not cause disease by increasing vesicular accumulation.
A previous report (Cooper et al., 2006) showed that the A53T
mutation caused a CPY trafficking block in yeast at earlier
time points than WT ?-synuclein. However, there was no
reported difference in toxicity between WT and A53T
?-synuclein expressing cells, suggesting that this CPY traf-
ficking defect may be independent of vesicular accumula-
tion and toxicity. Alternatively, the difference in CPY traf-
ficking between WT and A53T ?-synuclein may be insufficient to
cause changes in the size and the number of vesicle clusters.
Because ?-syn fibrils are not detected in ?-syn–expressing
yeast cells, our data are consistent with enhanced fibril
formation due to the A53T mutation being downstream
from vesicle accumulations. Finally, these yeast models of
synucleinopathies may represent unique systems to study
?-syn disruption of cellular trafficking and toxicity in PD
that is independent from ?-syn fibril formation.
Although ER-Golgi transport-vesicle markers accumulate
in yeast based on the colocalization of ?-syn-EGFP with
Ypt1 (Cooper et al., 2006), our EM data suggest that there
may be several other types of vesicles in the ?-syn-EGFP
vesicular inclusions, because vesicles of various sizes are
observed. For example, we identified Sec4p, a secretory
vesicle associated Rab GTPase within ?-syn-EGFP accumu-
lations, suggesting that secretory vesicles are also present.
Thus, our data suggest that the ?-syn-EGFP accumulations
include vesicular accumulations composed of at least two
different types of vesicles in the transport and secretion
pathway and that ?-syn is able to disrupt vesicular organi-
zation at two stages of the transport/secretion pathway.
However, it cannot be ruled out that the markers themselves
are mislocalized and that these are not true secretory trans-
port vesicles. This observation may reflect an exaggeration
of the normal function of ?-syn, which has been hypothe-
sized to play a role in the organization and recycling of
synaptic vesicles. When ?-syn is overexpressed or misregu-
lated in humans, it may disrupt these pathways. However,
in yeast there are no synaptic vesicles and ?-syn may per-
form similar functions involving other types of vesicles,
including secretory vesicles and ER-Golgi transport vesicles.
?-Synuclein-induced Vesicular Accumulation
Vol. 19, March 20081101
?-syn–induced vesicular accumulations, as demonstrated
here in yeast, may represent an important early step in the
pathogenesis of PD. Indeed, dense accumulations of vesicles
concentrated in the periphery of LBs were observed in neu-
rons in the substantia nigra of a PD patient. These vesicles
may be the remains of larger vesicular accumulations that
were caused by vesicle binding of ?-syn as seen in the yeast
model. These vesicular structures may provide a scaffold for
?-syn fibrillization and therefore may be directly involved in
the formation of pathological LBs. Although examination of
additional cases is required to verify these findings, vesicu-
lar accumulations have been observed in neuronal pale
body-like structures that have been speculated to be precur-
sors of LBs (Hayashida et al., 1993). Thus, accumulation of
?-syn–associated vesicles may result in the eventual forma-
tion of fibrillar ?-syn as LBs, which compromise neuronal
survival. Alternatively, the accumulation of vesicles may
directly result in neuronal toxicity. Further examination of
cell and animal models is needed to explore these hypothe-
In conclusion, we have demonstrated that the inclusions
seen in S. cerevisiae expressing ?-syn are comprised of clus-
ters of vesicles. Although vesicular accumulations can be
detected in LB-containing neurons in human PD, ?-syn
amyloid fibrils are present in authentic LBs but not in yeast
cells, suggesting that vesicular clustering may be an early
step in the pathogenesis of LB. Future studies in this and
other model systems will provide additional insight into the
exact relationship between this vesicular accumulation phe-
notype and ?-syn fibril formation in human neurodegenera-
We thank Dr. Kelvin Luk for his critical reading of our manuscript and Dr.
Hiro Uryu (University of Pennsylvania) for providing the human PD case
material. This work was supported by grants from the National Institutes of
Health (AG-P01-09215, GM T32 07229) and the Picower Foundation. V.M.-
Y.L. is the John H. Ware III professor in Alzheimer’s disease research. J.Q.T.
is the William Maul Measey-Truman G. Schnabel, Jr. Chair of Geriatric
Medicine and Gerontology. We acknowledge the Penn Bio-Imaging Core for
electron microscopy, and we thank the families of the patients used in this
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