NeuralActivityControlstheSynapticAccumulation of-Synuclein

Full text

Neurobiology of Disease
Neural Activity Controls the Synaptic Accumulation
of
␣
-Synuclein
Doris L. Fortin,
1
Venu M. Nemani,
1
Susan M. Voglmaier,
1
Malcolm D. Anthony,
1
Timothy A. Ryan,
2
and
Robert H. Edwards
1
1
Departments of Neurology and Physiology, Graduate Programs in Biomedical Sciences, Cell Biology, and Neuroscience, University of California, San
Francisco, San Francisco, California 94143-2140, and
2
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021
The presynaptic protein
␣
-synuclein has a central role in Parkinson’s disease (PD). However, the mechanism by which the protein
contributes to neurodegeneration and its normal function remain unknown.
␣
-Synuclein localizes to the nerve terminal and interacts
with artificial membranes in vitro but binds weakly to native brain membranes. To characterize the membrane association of
␣
-synuclein
in living neurons, we used fluorescence recovery after photobleaching. Despite its enrichment at the synapse,
␣
-synuclein is highly
mobile, with rapid exchange between adjacent synapses. In addition, we find that
␣
-synuclein disperses from the nerve terminal in
response to neural activity. Dispersion depends on exocytosis, but unlike other synaptic vesicle proteins,
␣
-synuclein dissociates from
the synaptic vesicle membrane after fusion. Furthermore, the dispersion of
␣
-synuclein is graded with respect to stimulus intensity.
Neural activity thus controls the normal function of
␣
-synuclein at the nerve terminal and may influence its role in PD.
Key words:
␣
-synuclein; membrane association; synaptic vesicle; neural activity; Parkinson’s disease; synapsin
Introduction
Genetic studies have implicated the protein
␣
-synuclein in the
pathogenesis of Parkinson’s disease (PD). Mutations in
␣
-synuclein cause a rare autosomal-dominant form of PD (Poly-
meropoulos et al., 1997; Kruger et al., 1998; Zarranz et al., 2004).
However, the protein also accumulates in Lewy bodies and dys-
trophic neurites of idiopathic PD, suggesting an important
pathogenic role for
␣
-synuclein even in the absence of inherited
mutations (Spillantini et al., 1998a,b; Galvin et al., 1999). The
recent demonstration that a simple increase in dosage of
the
␣
-synuclein gene can cause PD further supports a role for the
wild-type protein in the idiopathic disorder (Singleton et al.,
2003). However, the circumstances that trigger a pathogenic role
for wild-type
␣
-synuclein and indeed its normal function remain
unclear.
Considerable work has suggested a role for
␣
-synuclein in
neurotransmitter release. Neurons deficient in
␣
-synuclein show
a reduction in the reserve pool of synaptic vesicles required for
the response to repeated stimulation (Murphy et al., 2000; Cabin
et al., 2002). Other studies using knock-out mice have not de-
tected changes in hippocampal synaptic physiology (Abeliovich
et al., 2000; Chandra et al., 2004) but rather, increases in dopa-
mine release (Abeliovich et al., 2000; Yavich et al., 2004).
Consistent with a role in neurotransmitter release,
␣
-synuclein localizes to the nerve terminal (Jakes et al., 1994; Iwai
et al., 1995; Withers et al., 1997). Originally identified as a result
of its association with synaptic vesicles (Maroteaux et al., 1988),
␣
-synuclein lacks a transmembrane domain or lipid anchor and
has been considered a peripheral membrane protein. Indeed,
␣
-synuclein binds to artificial membranes in vitro by adopting an
␣
-helical conformation (Davidson et al., 1998; Jo et al., 2000;
Eliezer et al., 2001; Chandra et al., 2003).
␣
-Synuclein also asso-
ciates with axonal transport vesicles, lipid droplets, and yeast
membranes (Jensen et al., 1998; Cole et al., 2002; Outeiro and
Lindquist, 2003). However,
␣
-synuclein behaves almost entirely
as a soluble protein in brain extracts (Maroteaux and Scheller,
1991; Jakes et al., 1994; Iwai et al., 1995), raising questions about
the mechanism of synaptic localization in vivo. We found recently
that an interaction with membrane microdomains known as
lipid rafts is required for the localization of
␣
-synuclein to the
nerve terminal (Fortin et al., 2004). The PD-associated A30P mu-
tation specifically disrupts both raft association and the synaptic
localization of
␣
-synuclein (Fortin et al., 2004). All of these stud-
ies have, however, relied on biochemical approaches or steady-
state localization in fixed cells, limiting the scope of analysis and
conclusions.
To characterize the kinetic behavior of
␣
-synuclein in a phys-
iological context, we used live cell imaging of green fluorescent
protein (GFP)-tagged proteins expressed in dissociated hip-
pocampal neurons. Fluorescence recovery after photobleaching
(FRAP) experiments show that the localization of
␣
-synuclein at
the nerve terminal is highly dynamic, with rapid exchange of the
protein between neighboring synapses. However, the mobility of
Received July 14, 2005; revised Sept. 30, 2005; accepted Oct. 7, 2005.
This work was supported by the Hillblom Foundation (D.L.F), the University of California, San Francisco Medical
Scientist Training Program grant from the National Institutes of Health (V.M.N.), the Howard Hughes Medical
Institute (S.M.V.), a Society for Neuroscience Minority Fellowship (M.D.A.), the Sandler Neurogenetics Program, and
the Valley Foundation (R.H.E.). We thank S. Finkbeiner and K. Svoboda for helpful discussion and W. Yan, C. Tran, J.
Park, and Y. Dobryy for technical assistance.
Correspondence should be addressed to R. H. Edwards, Departments of Neurology and Physiology, University of
California, San Francisco School of Medicine, 600 16th Street, San Francisco, CA 94143-2140. E-mail:
edwards@itsa.ucsf.edu.
DOI:10.1523/JNEUROSCI.2922-05.2005
Copyright © 2005 Society for Neuroscience 0270-6474/05/2510913-09$15.00/0
The Journal of Neuroscience, November 23, 2005 •25(47):10913–10921 • 10913

wild-type
␣
-synuclein is distinctly slower
than that of GFP alone and the PD-
associated A30P mutant, which is not en-
riched at the nerve terminal (Fortin et al.,
2004). The slowed recovery of wild-type
␣
-synuclein indicates that transient, rap-
idly reversible interactions account for the
steady-state localization of
␣
-synuclein to
the synapse. We also find that neural activ-
ity results in the graded dispersion of
␣
-synuclein from synapses, and dispersion
requires synaptic vesicle exocytosis, not
simply calcium entry. Unlike other synap-
tic vesicle proteins (Sankaranarayanan
and Ryan, 2000; Li and Murthy, 2001),
however,
␣
-synuclein does not accumu-
late in the perisynaptic region after mem-
brane fusion, indicating that it dissociates
from synaptic membranes after exocyto-
sis. Thus, neural activity controls both the
synaptic localization and membrane asso-
ciation of
␣
-synuclein.
Materials and Methods
Plasmids. The plasmid encoding GFP–synaptic
vesicle protein 2 (GFP–SV2) was the generous
gift from C. Waites (Stanford University, Palo
Alto, CA). Subcloning of wild-type and mutant
␣
-synuclein as well as synapsin I into enhanced GFP vectors (Clontech,
Palo Alto, CA) has been described previously (Chi et al., 2001; Fortin et
al., 2004).
Hippocampal cultures and transfections. Dissociated hippocampal cul-
tures containing glia were prepared from embryonic day 18.5 rats, plated
on glass coated with 50
␮
g/ml poly-D-lysine, and maintained in Neuro-
basal medium supplemented with B27, Glutamax I (Invitrogen, San Di-
ego, CA), penicillin, and streptomycin (Higgins and Banker, 1998). Hip-
pocampal neurons were transfected at 7 d in vitro using Effectene
(Qiagen, Valencia, CA). One-half of the culture medium was replaced at
the time of transfection and then 1 week later. Cells were imaged at 15–21
din vitro. Where indicated, neurons were treated with 10 nMtetanus
toxin (List Biologicals, Campbell, CA) for 16 h before imaging and with 5
␮
Mepoxomicin for 15 h before imaging (Ehlers, 2003).
Immunofluorescence. At 15–21 d in vitro, neuronal cultures were fixed
in PBS containing 4% paraformaldehyde. Cells were permeabilized,
blocked, and stained in PBS containing 0.05% saponin, 5% cosmic calf
serum (Hyclone, Logan, UT) with antibodies to
␣
-synuclein (BD Bio-
sciences, San Diego, CA), synapsin I (Synaptic Systems, Goettingen, Ger-
many), and vesicular glutamate transporter 1 (VGLUT1) (Chemicon,
Temecula, CA). Secondary antibodies conjugated to FITC, cyanine 3
(Cy3), or Cy5 were obtained from Jackson ImmunoResearch (West
Grove, PA). Fluorescence was visualized and images acquired on a Zeiss
(Oberkochen, Germany) LSM 510 confocal microscope using a 63⫻oil
objective [numerical aperture (NA), 1.4].
Fluorescence recovery after photobleaching. Neurons were imaged at
18 –21 d in vitro with a Zeiss LSM510 confocal microscope and a 63⫻oil
immersion objective (NA, 1.4) in Tyrode’s solution (in mM: 119 NaCl,
2.5 KCl, 2 CaCl
2
, 2 MgCl
2
, 10 HEPES-NaOH, pH 7.4, 30 glucose) con-
taining 10
␮
MCNQX and 50
␮
MAP-5. Physiological temperature was
maintained at 37°C on a heated stage, and individual cultures were im-
aged for ⱕ2 consecutive hours. Transfected neurons were identified un-
der epifluorescence and imaged using the 488 nm line of the argon laser.
Emitted light was collected using a 505 long-pass filter. To increase the
depth of field and collect light from the entire synapse, the pinhole was set
to 2.5
␮
m. During image collection, laser power was maintained at
0.3–2% to reduce photobleaching. After collecting the first image, laser
power was increased to 100% and a single synapse bleached by five con-
secutive scans. Laser power was then restored to 0.3–2%, and 40 images
were collected every 400 ms. Fluorescence in the bleached area was quan-
tified using the Zeiss LSM510 software and converted to relative fluores-
cence, with the initial fluorescence defined as 100% and that after bleach-
ing as 0%. Data are shown are mean ⫾SEM.
Electrical stimulation and epifluorescence microscopy. Transfected neu-
rons were imaged at 15–21 d in vitro with an inverted epifluorescence
Nikon (Tokyo, Japan) microscope and 60⫻oil objective (NA, 1.4) at
room temperature in a closed, laminar flow chamber containing plati-
num electrodes (Warner Instruments, Hamden, CT). Action potentials
were elicited using an A310 Accupulser (World Precision Instruments,
Sarasota, FL) at 10 Hz with 1 ms bipolar current pulses yielding fields of
5–10 V/cm across the chamber after acquisition of the fifth image. Sam-
ples were illuminated using a Xenon lamp (Sutter Instruments, Novato,
CA) with a 492/18 nm excitation filter and 513–547 nm bandpass emis-
sion filter (Chroma, Rockingham, VT), and images were acquired every
6 s on a CCD camera (Hamamatsu, Bridgewater, NJ) using on-chip 2 ⫻
2 pixel binning. Stimulation, filter wheels, shutter, and camera were con-
trolled by MetaMorph software (Universal Imaging, Downington, PA).
Fluorescence in 4 ⫻4 pixels regions, manually placed on the center of
individual synapses, was quantified, baseline values averaged from the
first five frames (before stimulation), and the dynamics of GFP-tagged
proteins expressed as fractional fluorescence change. Data shown are
mean ⫾SEM.
Results
Transient, rapidly reversible interactions localize
␣
-synuclein
to the synapse
At steady-state,
␣
-synuclein exhibits synaptic enrichment similar
in extent to that observed for polytopic membrane proteins of the
synaptic vesicle such as VGLUT1 (Fig. 1A) (Withers et al., 1997;
Fortin et al., 2004; Specht et al., 2005). To understand how this
protein, which does not contain a transmembrane domain or
known lipid anchor, localizes to the nerve terminal, we studied
the dynamics of
␣
-synuclein in live hippocampal neurons. Trans-
fection of hippocampal neurons with human
␣
-synuclein fused
at its N terminus to enhanced GFP (GFP–
␣
-synuclein) leads to a
modest ⬃50% overexpression compared with untransfected cells
as determined by quantitative immunofluorescence using an an-
Figure 1. Steady-state enrichment of
␣
-synuclein at the synapse. A, Endogenous
␣
-synuclein colocalizes with VGLUT1 at the
synapses of cultured hippocampal neurons. In the overlay images,
␣
-synuclein and VGLUT1 are pseudocolored green and red,
respectively. Colocalized
␣
-synuclein and VGLUT1 appear yellow in the overlay. Scale bars: top, 20
␮
m; bottom, 4
␮
m. B,
GFP–
␣
-synuclein (GFP-
␣
syn) localizes to the synapses of transfected rat hippocampal neurons. Transfection leads to a modest
⬃50%increaseintheexpressionof total
␣
-synuclein(synuclein)detectedusinganantibody that recognizes both rat and human
proteins.GFP–
␣
-synucleinispseudocoloredgreen in the overlay, whereas total
␣
-synuclein(transfectedandendogenous) is red.
Scale bar, 6
␮
m.
10914 •J. Neurosci., November 23, 2005 •25(47):10913–10921 Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein

tibody that recognizes both rat and human proteins: 1545 ⫾39
arbitrary fluorescence units (AFU) in transfected cells (n⫽320
boutons from eight different neurons) versus 1050 ⫾21 AFU in
untransfected cells (n⫽409 boutons from the same eight fields).
This level of overexpression does not perturb the enrichment of
either human or endogenous rat
␣
-synuclein at the nerve termi-
nal of transfected neurons (Fig. 1B). In addition, the overexpres-
sion does not appear to affect the development of transfected
neurons or the localization of other presynaptic proteins includ-
ing synaptophysin, SV2, synapsin, and VGLUT1 (data not
shown). The synaptic localization of GFP–
␣
-synuclein also
strongly suggests that the GFP fusion does not influence the be-
havior of
␣
-synuclein or its interactions at the nerve terminal.
Other N-terminal tags such as glutathione S-transferase or the
calmodulin-binding protein CBP do not interfere with the mem-
brane binding of
␣
-synuclein in vitro and do not change the
requirement for acidic phospholipid headgroups or the
␣
-helical
transition of synuclein during membrane binding (Perrin et al.,
2000; Kubo et al., 2005). Although we cannot completely exclude
some difference from the untagged protein that has eluded detec-
tion in vitro and in vivo, GFP–
␣
-synuclein thus appears to serve as
an accurate, nondisruptive reporter for the endogenous protein.
To assess the mobility of
␣
-synuclein in live cells, we used
FRAP (Lippincott-Schwartz et al., 2001). After photobleaching
GFP–
␣
-synuclein at a single synapse, we observed rapid fluores-
cence recovery in the bleached region (Fig. 2A), suggesting that
wild-type human
␣
-synuclein is highly mobile in hippocampal
neurons, an unexpected finding given its strong synaptic enrich-
ment. No fluorescence recovery was observed after photobleach-
ing fixed neurons, confirming that the conditions used here irre-
versibly bleach GFP–
␣
-synuclein and that recovery results from
the movement of unbleached molecules into the bleached area
(data not shown). In addition, fluorescence recovery at the
bleached synapse was typically accompanied by a decrease in flu-
orescence at neighboring boutons (24.3 ⫾0.8%; n⫽57 bou-
tons), consistent with adjacent synapses acting as the source of
mobilized GFP–
␣
-synuclein (Fig. 2A). Recovery after photo-
bleaching was best fit with a single exponential, supporting the
behavior of GFP–
␣
-synuclein as a homogeneous population.
GFP–
␣
-synuclein thus exchanges rapidly between neighboring
synapses.
Rapid recovery after photobleaching makes it unlikely that
␣
-synuclein remains tightly associated with synaptic vesicles.
Synaptic vesicles can exhibit mobility within a single terminal
under certain conditions (Henkel et al., 1996), but there is no
precedent for their movement between terminals at rest (Henkel
et al., 1996; Kraszewski et al., 1996). However, spontaneous, sus-
tained network activity can lead to the mixing of synaptic vesicles
between terminals (Li and Murthy, 2001). To exclude activity-
dependent vesicle mobility as a cause for the rapid recovery of
␣
-synuclein, we used the glutamate receptor antagonists CNQX
and AP-V in the FRAP experiments described above. In addition,
we measured the recovery after photobleaching of a GFP fusion
to SV2, an integral membrane protein of synaptic vesicles (Lowe
et al., 1988). The fluorescence of GFP–SV2 does not recover from
Figure 3. GFP–
␣
-synuclein has no immobile fraction. A, To assess a possible immobile
fraction of
␣
-synuclein, individual boutons were bleached consecutively, and the extent of
recovery normalized to the fluorescence observed at the start of each bleach. The extent of
fluorescence recovery for GFP–
␣
-synuclein (
␣
syn) remains the same after the second bleach,
excluding an immobile fraction. In contrast, the recovery of synapsin (syp) increases after the
second bleach, consistent with an immobile fraction. B, The extent of recovery for GFP–
␣
-
synuclein after bleach 1 and 2 (filled) falls on a line with slope ⬃1, confirming a similar extent
of recovery after both bleaches and no immobile fraction. In the case of synapsin (open), how-
ever, the ratio of recovery exceeds 1, indicating a larger recovery after the second bleach and
hence an immobile fraction.
Figure 2.
␣
-Synuclein interacts transiently with synaptic components. A, A single, isolated hippocampal bouton expressing GFP–
␣
-synuclein was photobleached at high laser power, and
recovery was monitored every 400 ms thereafter. GFP–
␣
-synuclein recovers rapidly after photobleaching. The two synapses outside the bleached box show a reduction in fluorescence during
recovery of the bleached region. The color scale is shown to the right. Scale bar, 2
␮
m. B, The intensity of fluorescence at the bleached synapse was quantified and expressed as a percentage of initial
fluorescence, with fluorescence after the bleach defined as zero. GFP (green) and GFP–A30P (blue) recover with similar, rapid kinetics after photobleaching, consistent with their behavior as soluble
proteins. In contrast, GFP–
␣
-synuclein (red) recovers more slowly, indicating a distinct but rapidly reversible interaction with synaptic components. GFP–synapsin (GFPsyp; purple) recovers more
slowly than
␣
-synuclein, consistent with a higher affinity for synaptic vesicles. GFP–SV2 (black) does not recover after photobleaching, indicating that synaptic vesicles do not exchange between
synapses, at least during this time scale. Data shown are averages ⫾SEM of 30 –40 boutons per construct.
Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein J. Neurosci., November 23, 2005 •25(47):10913–10921 • 10915

photobleaching in the time course of these
experiments (Fig. 2B), consistent with the
relative immobility of synaptic vesicles.
The rapid recovery of
␣
-synuclein after
FRAP thus excludes the tight association
of
␣
-synuclein with synaptic vesicles. A fu-
sion of GFP to synapsin I, another periph-
eral membrane protein associated with syn-
aptic vesicles, also recovers more slowly from
photobleaching than
␣
-synuclein. The
FRAP recovery kinetics thus indicates that
␣
-synuclein binds more weakly to synaptic
vesicles than synapsin (Fig. 2B) (Phair and
Misteli, 2001).
Despite its rapid recovery after photo-
bleaching, wild-type
␣
-synuclein recovers
more slowly than GFP alone. The slower
recovery of GFP–
␣
-synuclein does not re-
flect its larger mass, because a fusion of
GFP to the PD-associated A30P
␣
-synuclein (GFP–A30P), which has es-
sentially the same molecular weight as
GFP–
␣
-synuclein, recovers as rapidly as
GFP alone (Fig. 2B). The recovery kinetics
of GFP–A30P is consistent with its lack of
synaptic enrichment and its behavior as a
soluble protein in neurons (Fortin et al.,
2004). The synaptic localization of wild-
type
␣
-synuclein thus reflects rapidly re-
versible interactions that enable exchange
of the protein between adjacent synapses.
Nonetheless, these transient interactions
are sufficient for the steady-state enrich-
ment of wild-type
␣
-synuclein at the nerve
terminal, and their disruption by the A30P
mutation eliminates synaptic localization.
To determine whether a small fraction
of
␣
-synuclein might be immobile, we
have assessed the extent as well as the rate
of recovery after photobleaching. GFP and
GFP–A30P do not recover fully after pho-
tobleaching, presumably reflecting the
limited local pool of protein from which
recovery can occur (Elson and Qian,
1989). The extent of their recovery (⬃80%)
was therefore considered the maximal re-
covery for a soluble protein. The reduced re-
covery of GFP–
␣
-synuclein (⬃70%) relative
to that of GFP and GFP–A30P suggests that a
small proportion (⬃10%) of the protein
may be immobile (Fig. 2B). To assess more
accurately the existence of an immobile frac-
tion, we repeatedly photobleached the same
synapse. In the case of a true immobile frac-
tion, a second photobleach will be followed
by more complete recovery than the first
photobleach, because the fluorescence is
normalized to that observed at the start
of each bleach, thereby eliminating the con-
tribution of the immobile fraction to recovery from the second
bleach (Lippincott-Schwartz et al., 2003). In the case of GFP–synap-
sin, recovery is substantially greater after the second photobleach
(Fig. 3), suggesting the presence of a significant immobile fraction. In
contrast, the fluorescence of GFP–
␣
-synuclein recovers to the same
extent after the first and second photobleach (Fig. 3), excluding a
significant immobile fraction and indicating that the entire pool of
protein is mobile and can exchange between adjacent synapses.
Figure 4.
␣
-Synuclein disperses in response to depolarization. A, Neurons were fixed (rest), depolarized with 45 mMKCl and imme-
diately fixed (stimulated), or depolarized followed by a 10 min recovery (recovery) before fixation. Synaptic boutons were identified by
VGLUT1 staining and are indicated with arrowheads. Similar to synapsin, endogenous
␣
-synuclein disperses from boutons after depolar-
ization. Unlike synapsin, however,
␣
-synuclein does not accumulate in the axon (arrows) after stimulation. Ten minutes after recovery,
synapsin has reclustered in the synaptic terminal and colocalizes with VGLUT1.
␣
-Synuclein does not reaccumulate at the synapse in this
timeframe.Scale bar, 2
␮
m.B,At rest, GFP–
␣
-synucleinisenriched at synaptic boutons and dispersesduringstimulation with 600 action
potentials(AP)delivered at 10Hz.Minimalreclustering of theproteinhas occurred by 5min(recovery). The color scaleisshown to the right.
Scale bar, 2
␮
m. C, No dispersion of
␣
-synuclein occurs during stimulation with 600 AP in calcium-free medium (gray). After stimulation
in the absence of calcium, the cells were washed in calcium-containing medium for 10 min and were then restimulated (red). The
dispersion of
␣
-synuclein thus depends on calcium entry. The traces are the average ⫾SEM dispersion at 30 synapses from one repre-
sentative cell. D, Average dispersion of GFP–
␣
-synuclein during sequential stimulation in calcium-free and calcium-containing medium.
Mediumwasexchangedduring a 10 min rest separating the two stimulationrounds.Errorbarsindicate SEM. p⬍0.0001, Student’s ttest;
n⫽138 synapses from three cells.
10916 •J. Neurosci., November 23, 2005 •25(47):10913–10921 Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein

Neural activity disperses
␣
-synuclein from synaptic boutons
Neural activity modulates the localization of multiple synaptic
proteins. For example, synapsin disperses from the synapse early
in the course of stimulation, suggesting dissociation from synap-
tic vesicles before their fusion with the plasma membrane (Chi et
al., 2001, 2003). Similarly, depolarization of hippocampal neu-
rons with high K
⫹
influences the distribution of
␣
-synuclein
observed in fixed cells. Before stimulation, endogenous
␣
-synuclein concentrates at synaptic boutons where it colocalizes
with synapsin and VGLUT1 (Fig. 4A, rest). Depolarization with
high K
⫹
results in the loss of
␣
-synuclein from synaptic boutons
(Fig. 4A, stimulated, arrowheads). Unlike synapsin, however,
␣
-synuclein does not increase substantially in the axon after stim-
ulation (Fig. 4A, stimulated, arrows), and synaptic
␣
-synuclein
does not recover within 10 min after cessation of the stimulus
(Fig. 4A, recovery, arrowheads).
To characterize the activity-dependent dispersion of
␣
-synuclein, we imaged the GFP fusion in living hippocampal
neurons. Similar to the endogenous protein, the amount of GFP–
␣
-synuclein at the synapse decreases by ⬃20% after a 600 action
potentials stimulus (Fig. 4B–D). GFP–
␣
-synuclein does not ap-
pear to accumulate in the axon, and very little returns to the
boutons after the stimulus has stopped (Fig. 4B,C). The loss of
synaptic GFP–
␣
-synuclein produced by activity depends on ex-
ternal calcium (Fig. 4C,D), indicating a requirement for calcium
entry. GFP and GFP–A30P show no change in fluorescence after
stimulation, consistent with their behavior as soluble proteins
and supporting the significance of the dispersion observed for
GFP–
␣
-synuclein (Fig. 5A). The PD-associated A53T mutant,
which localizes normally to the nerve terminal (Fortin et al.,
2004), exhibits the same activity-dependent dispersion as wild-
type
␣
-synuclein (Fig. 5B).
The kinetics of dispersion differs for the two peripheral mem-
brane proteins synapsin and
␣
-synuclein (Fig. 5A). The activity-
dependent dispersion of
␣
-synuclein appears somewhat slower than
that of synapsin (Fig. 5A) (Chi et al., 2001).
␣
-Synuclein may thus
disperse at a later step during the evoked release of neurotransmitter.
In addition, the synaptic fluorescence of GFP–synapsin rapidly re-
covers to prestimulation levels, whereas GFP–
␣
-synuclein recov-
ers only in part, resulting in the net loss of
GFP–
␣
-synuclein from the nerve terminal
(Fig. 5A).
Exocytosis triggers the dispersion
of
␣
-synuclein
What triggers the dispersion of
␣
-synuc-
lein from the nerve terminal? The strin-
gent requirement for calcium indicates
that dispersion of
␣
-synuclein occurs after
action potential invasion in the terminal
(Fig. 4C,D). The dispersion of synapsin
also requires calcium entry and results
from phosphorylation by calcium/calmo-
dulin-dependent protein kinase II before
exocytosis (Chi et al., 2001). However, the
delay in movement of
␣
-synuclein relative
to synapsin suggests that
␣
-synuclein may
disperse at a later step in the exocytic pro-
cess. We therefore assessed dispersion of
␣
-synuclein in neurons pretreated with
tetanus toxin, which blocks the regulated
exocytosis of synaptic vesicles without af-
fecting calcium influx. Because the disper-
sion of synaptic vesicle proteins requires fusion of synaptic vesi-
cles with the plasma membrane and subsequent diffusion along
the surface of the axon (Sankaranarayanan and Ryan, 2000; Li
and Murthy, 2001), tetanus toxin substantially reduces the dis-
persal of GFP–SV2 (Fig. 6A). Tetanus toxin treatment also im-
pairs the dispersion of
␣
-synuclein (Fig. 6A), indicating that
the exocytic event itself triggers the movement of
␣
-synuclein.
The similar dependence on exocytosis presumably accounts
for the similar kinetics of SV2 and
␣
-synuclein dispersion from
the synapse (Fig. 5A). In contrast, the dispersion of synapsin is
unaffected by tetanus toxin (Fig. 6A), consistent with its release
from synaptic vesicles before exocytosis (Chi et al., 2001).
The extent of synaptic vesicles exocytosis, the time course of
endocytosis, and the reclustering of synaptic vesicles are regu-
lated by the duration of a stimulus (Sankaranarayanan and Ryan,
2000; Li and Murthy, 2001). To determine whether the extent of
␣
-synuclein dispersion from the terminal or the time course of
recovery also depends on stimulus duration, we varied the length
of the 10 Hz pulse, in effect delivering different numbers of action
potentials. Stimulation with a larger number of action potentials
increases the average extent of
␣
-synuclein dispersion from syn-
aptic boutons (Fig. 6B). This may reflect the increasing probabil-
ity of all-or-none dispersion from individual boutons. Alterna-
tively, the extent of
␣
-synuclein dispersion at individual boutons
may be graded, reflecting a fixed amount of dispersion per action
potential. Figure 6Cindeed shows that the dispersion of
␣
-synuclein within individual terminals is graded rather than
bimodal. The amount of
␣
-synuclein at the nerve terminal thus
reflects the level of previous synaptic activity.
␣
-Synuclein dissociates from the synaptic vesicle membrane
after fusion
Where does
␣
-synuclein go after exocytosis? The failure to ob-
serve a significant increase in axonal
␣
-synuclein after stimula-
tion raised the possibility that the protein is degraded in an
activity-dependent manner. However, Western analysis showed
that the total amount of
␣
-synuclein does not change after depo-
larization of untransfected neurons (data not shown). Arguing
further against a role for protein degradation, pretreatment of
Figure 5. Synaptic vesicle proteins disperse with different kinetics after stimulation. A, GFP–
␣
-synuclein (GFP
␣
syn) and
GFP–A53T–
␣
-synuclein (data not shown) disperse more slowly than GFP–synapsin (GFP-syp) after 600 action potentials (AP)
delivered at 10 Hz. In contrast, GFP and GFP–A30P–
␣
-synuclein (GFPA30P) do not disperse after 600 AP, consistent with their
behaviorassolubleproteins.Thekineticsof
␣
-synucleindispersionmostcloselyresemblesthatofGFP–SV2. The traces shown are
averages ⫾SEM of 22–54 boutons from one representative cell for each construct. B, Average maximal dispersion of GFP-tagged
proteins immediately after 600 AP. Error bars indicate SEM (n⬎300 boutons from 6 –9 cells for each construct).
Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein J. Neurosci., November 23, 2005 •25(47):10913–10921 • 10917

neurons with the proteasome inhibitor ep-
oxomicin does not affect the activity-
dependent loss of GFP–
␣
-synuclein from
the nerve terminal. Treated boutons show
a decrease in fluorescence of 24.8 ⫾1.1%
(⌬F/F
0
) after 600 action potentials (n⫽
107 boutons from three cells), in the same
range as the decrease in fluorescence ob-
served in untreated cells (Figs. 4, 5).
Over the entire field, quantification of
GFP–
␣
-synuclein fluorescence indicates a
modest 1.9 ⫾0.4% reduction in fluores-
cence (Fig. 7B), similar in extent to that
observed for stimulation of neurons ex-
pressing GFP and GFP–A30P (6.4 ⫾0.4
and 6.6 ⫾0.5% decrease, respectively) but
significantly smaller than the stimulation-
induced dispersion of GFP–
␣
-synuclein
observed at boutons (18.3 ⫾0.5% de-
crease) (Figs. 4, 7). Thus, the total amount
of GFP–
␣
-synuclein does not change with
stimulation, supporting rapid diffusion
away from the synapse rather than degra-
dation or secretion.
Because the dispersion of
␣
-synuclein
requires synaptic vesicle exocytosis and
follows kinetics similar to that of SV2,
␣
-synuclein may behave like a typical
membrane protein of the synaptic vesicle,
inserting into the plasma membrane after
exocytosis and spreading laterally into the
perisynaptic area before recycling into
newly formed synaptic vesicles that even-
tually recluster in the bouton (Sankarana-
rayanan and Ryan, 2000; Li and Murthy,
2001). During a train of 600 action poten-
tials, the fluorescence of both SV2 and
␣
-synuclein in the center of the bouton
declines to a similar extent (21.6 ⫾0.9%
decrease for SV2, n⫽105 boutons from
three cells; 24.3 ⫾0.8% decrease for
␣
-synuclein, n⫽163 boutons from four
cells) and with similar kinetics (Fig. 7A). In
the case of SV2, this decrease in fluores-
cence over the bouton is accompanied by
an equivalent increase in fluorescence over
the perisynaptic region (13.5 ⫾0.9% in-
crease for each of the two perisynaptic ar-
eas surrounding a bouton; n⫽184 peri-
synaptic areas from three cells) (Fig. 7A). Synaptic vesicle
recycling then redistributes the accumulated perisynaptic fluo-
rescence back to the center of the bouton (Fig. 7A). In contrast,
the activity-dependent dispersion of
␣
-synuclein is not accompa-
nied by an equivalent accumulation in the perisynaptic area
(0.38 ⫾0.52% increase; n⫽308 perisynaptic areas from four
cells), suggesting that the protein does not remain associated with
the plasma membrane after synaptic vesicle exocytosis (Fig. 7A).
Rather,
␣
-synuclein appears to dissociate from its binding site
after exocytosis, behaving like a soluble protein that rapidly dif-
fuses away from the bouton. We regularly observed a transient,
small, stimulation-induced increase in the perisynaptic fluores-
cence of
␣
-synuclein (Fig. 7A), presumably reflecting the rapid
movement of soluble
␣
-synuclein into the axonal cytoplasm.
Discussion
␣
-Synuclein exhibits enrichment at the nerve terminal equivalent
to that of an integral membrane protein of synaptic vesicles, but
the kinetic analysis in live neurons using FRAP indicates that it is
extremely mobile, exchanging rapidly between adjacent synapses.
We cannot detect even a small fraction of immobile
␣
-synuclein
by sequential photobleaching, whereas the peripheral membrane
protein synapsin exhibits a significant immobile fraction. None-
theless, the recovery of wild-type
␣
-synuclein from photobleach-
ing is slower than that of the A30P mutant. Because A30P–
␣
-
synuclein behaves as a soluble protein not enriched at the nerve
terminal (Fortin et al., 2004), we conclude that the slowed recov-
ery of wild-type
␣
-synuclein reflects the interactions that under-
Figure 6. The dispersion of
␣
-synuclein depends on exocytosis and shows a graded response to increasing numbers of action
potentials (AP). A, The average maximal dispersion after 600 AP in cells expressing GFP–
␣
-synuclein (GFP
␣
syn), GFPSV2, or
GFP–synapsin (GFPsyp) either with or without tetanus toxin pretreatment. Tetanus toxin greatly reduces the activity-dependent
dispersion of
␣
-synuclein and GFP–SV2. In contrast, tetanus toxin does not affect the dispersion of synapsin, consistent with its
dissociation before synaptic vesicle exocytosis. Error bars indicate SEM. *p⬍0.0001, Student’s ttest; n⬎140 boutons from four
to nine cells for each construct and treatment. B, Average maximal dispersion of
␣
-synuclein increases with the number of action
potentials, all delivered at 10 Hz. Error bars indicate SEM (n⬎80 boutons from 3–5 cells for each of the different stimulus
durations). C, Frequency histograms of GFP–
␣
-synuclein dispersion. The number of boutons exhibiting different levels of disper-
sion at the end of the stimulus is plotted as a function of initial fluorescence binned in 5% increments. Increasing AP number
produces greater dispersion, and the distributions show a graded rather than all-or-none, bimodal response.
10918 •J. Neurosci., November 23, 2005 •25(47):10913–10921 Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein

lie its synaptic localization. Thus, transient interactions are suffi-
cient for the precise steady-state localization of
␣
-synuclein to the
nerve terminal.
Live cell imaging enables us to reconcile previous conflicting
reports about the membrane interactions of
␣
-synuclein.
␣
-Synuclein binds to a number of artificial membranes in vitro,in
particular those containing phospholipids with an acidic head
group (Davidson et al., 1998; Jo et al., 2000; Eliezer et al., 2001).
However,
␣
-synuclein shows very little if any membrane associ-
ation in native brain extracts (Maroteaux and Scheller, 1991;
Jakes et al., 1994; Iwai et al., 1995). Although we cannot exclude
some difference between the behavior of GFP–
␣
-synuclein and
the untagged protein, our demonstration that GFP–
␣
-synuclein
exhibits only transient interactions in live neurons is consistent
with these observations. Presumably, the conditions used in vitro,
for example the absence of other protein, drive the binding of
␣
-synuclein to artificial membranes, whereas the high concentra-
tion of other cytoplasmic proteins present in cells make these
interactions transient and difficult to preserve during biochemi-
cal fractionation of native extracts. In contrast, FRAP experi-
ments can detect low-affinity interactions that mediate the syn-
aptic localization of
␣
-synuclein in vivo.
Can transient interactions account for the observed enrich-
ment of
␣
-synuclein at the synapse? Rapid association and disso-
ciation has been demonstrated previously to underlie the steady-
state localization of histones to stable compartments within the
nucleus (Phair and Misteli, 2000). Low-affinity interactions be-
tween cytoplasmic domains of proteins are also sufficient to form
and maintain stable compartments of the endoplasmic reticulum
(Snapp et al., 2003). Thus, readily reversible, low-affinity inter-
actions can create apparently stable cellular structures. However,
they also confer potential for rapid regulation. Indeed, we find
that neural activity regulates the synaptic localization of
␣
-synuclein.
Although several synaptic proteins have been reported to re-
distribute into the axon in response to stimulation, our results
show that the behavior of
␣
-synuclein is unique. The activity-
dependent dispersion of
␣
-synuclein is slower than that of an-
other peripheral membrane protein, synapsin. Although calcium
entry is required for the dispersion of both
␣
-synuclein and syn-
apsin, tetanus toxin only blocks the dispersion of
␣
-synuclein.
Thus, in contrast to synapsin, which disperses after calcium entry
but before synaptic vesicle fusion (Chi et al., 2001),
␣
-synuclein
disperses during or after fusion. Integral membrane proteins of
the synaptic vesicle such as synaptobrevin (Sankaranarayanan
and Ryan, 2000; Li and Murthy, 2001) and SV2 (this study) also
disperse after exocytosis, diffusing along the axonal membrane,
but unlike these proteins,
␣
-synuclein does not accumulate in the
perisynaptic region or the axon. Rather, the disappearance of
␣
-synuclein from the bouton and its absence from the perisyn-
aptic region indicate that it dissociates from the membrane after
fusion and rapidly diffuses away. Indeed, stimulation does not
alter the localization of GFP or A30P–
␣
-synuclein, confirming
that dispersion of
␣
-synuclein involves a disruption of mem-
brane binding.
Unlike synapsin and integral synaptic vesicle membrane pro-
teins,
␣
-synuclein does not reaccumulate at the nerve terminal
within 10 min after stimulation. Synaptic vesicles reform within
at least 20 – 60 s after exocytosis (Gandhi and Stevens, 2003;
Fernandez-Alfonso and Ryan, 2004), indicating that their disap-
pearance cannot account for the failure to recover synaptic
␣
-synuclein. Interestingly,
␣
-synuclein is considered to behave as
a natively unfolded protein in solution (Weinreb et al., 1996) and
may not require ubiquitination for degradation by the protea-
some (Tofaris et al., 2001; Snyder et al., 2003), raising the possi-
bility that rapid proteolysis accounts for the disappearance of
synaptic
␣
-synuclein after stimulation. However, we cannot de-
tect a decrease in the level of the endogenous protein by Western
analysis after neuronal stimulation. In addition, inhibition of the
proteasome does not block the stimulation-induced dispersion
of GFP–
␣
-synuclein or promote its reclustering after stimula-
tion. Furthermore, the analysis of total GFP–
␣
-synuclein fluores-
cence (over axons as well as boutons) shows no significant decline
with stimulation, indicating that the loss of
␣
-synuclein from
synapses is balanced by its appearance elsewhere. Thus, the lack
of recovery after stimulation presumably reflects rapid diffusion
to distant sites. The low affinity of
␣
-synuclein for synaptic com-
ponents may contribute to this relatively unimpeded diffusion
and also to the lack of recovery. In addition, we do not know
whether
␣
-synuclein remains
␣
-helical or unfolds after dissocia-
Figure 7.
␣
-Synuclein dissociates from synaptic vesicles during exocytosis. A, The activity-
dependent dispersion of GFP–SV2 (SV2; filled squares) from synaptic boutons is accompanied
by an increase in fluorescence in the perisynaptic area (filled circles). After endocytosis and the
reclustering of synaptic vesicles, the fluorescence of GFP-SV2 returns to the center of the bou-
ton. In contrast, GFP–
␣
-synuclein (
␣
syn) does not accumulate perisynaptically (open circles)
during dispersion from the bouton (open squares), consistent with its dissociation from the
membrane after synaptic vesicle exocytosis. A small, brief increase in perisynaptic fluorescence
follows the onset of stimulation and may reflect the rapid diffusion of GFP–
␣
-synuclein away
from the bouton. The inset indicates the regions used for quantification of fluorescence at the
center of the bouton and in the perisynaptic area. The traces are averages ⫾SEM from one
representativecellforeachconstruct(n⫽30boutonsforSV2and75boutons for
␣
-synuclein).
B, Average integrated fluorescence intensity of GFP–
␣
-synuclein over the entire field declines
by 1.8 ⫾0.4% after 600 action potentials (AP). Please note the difference in scale from A. The
trace shows average ⫾SEM fluorescence (n⫽5 fields).
Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein J. Neurosci., November 23, 2005 •25(47):10913–10921 • 10919

tion from the membrane, and this may influence its ability to
rebind synaptic vesicles and hence to recluster.
We have observed the dispersion of
␣
-synuclein from the
bouton after strong stimulation (600 action potentials). How-
ever, smaller numbers of action potentials also produce easily
detectable dispersion. The loss of
␣
-synuclein from the terminal
thus correlates with the extent of stimulation, suggesting a phys-
iological role for the activity-dependent dispersion. Consistent
with this,
␣
-synuclein inhibits refilling of the readily releasable
synaptic vesicle pool, and this effect diminishes with repeated
stimulation (Abeliovich et al., 2000; Yavich et al., 2004). In par-
ticular,
␣
-synuclein knock-out mice exhibit immediate, strong
facilitation in response to high-frequency stimulation that does
not appear in wild-type animals until after one or two previous
trains of 600 action potentials each (Yavich et al., 2004). Because
we have demonstrated that this amount of stimulation is suffi-
cient to disperse
␣
-synuclein, the observation that wild-type an-
imals begin to behave like
␣
-synuclein knock-outs after stimula-
tion may simply reflect the loss of
␣
-synuclein from wild-type
terminals. The effects of
␣
-synuclein on transmitter release pre-
sumably reflect the amount of protein remaining at the nerve
terminal.
Activity may influence the role of
␣
-synuclein in PD. Because
␣
-synuclein has a central role in PD but inherited mutations are
rare, activity-dependent changes in
␣
-synuclein have the poten-
tial to influence triggering of the sporadic disorder. For example,
membrane association has been reported to modulate the ten-
dency of
␣
-synuclein to aggregate (Narayanan and Scarlata, 2001;
Perrin et al., 2001; Cole et al., 2002; Lee et al., 2002). The dissoci-
ation of
␣
-synuclein from the membrane after exocytosis may
thus regulate its aggregation and toxicity. Interestingly, mice
lacking the DJ-1 gene implicated in familial PD exhibit a defect in
dopamine release (Goldberg et al., 2005), which may influence
the predisposition to degeneration by affecting the dispersion of
␣
-synuclein. In addition, smoking protects against PD, presum-
ably through the action of nicotine (Gorell et al., 1999; Tanner et
al., 2002; Quik, 2004). Nicotine activates ionotropic receptors at
the nerve terminal to cause the exocytic release of dopamine
(McGehee et al., 1995; Zhou et al., 2001). Thus, nicotine may
protect against PD by causing the dispersion of
␣
-synuclein.
References
Abeliovich A, Schmitz Y, Farinas I, Choi-Lundberg D, Ho WH, Castillo PE,
Shinsky N, Verdugo JM, Armanini M, Ryan A, Hynes M, Phillips H,
Sulzer D, Rosenthal A (2000) Mice lacking alpha-synuclein display
functional deficits in the nigrostriatal dopamine system. Neuron
25:239 –252.
Cabin DE, Shimazu K, Murphy D, Cole NB, Gottschalk W, McIlwain KL,
Orrison B, Chen A, Ellis CE, Paylor R, Lu B, Nussbaum RL (2002) Syn-
aptic vesicle depletion correlates with attenuated synaptic responses to
prolonged repetitive stimulation in mice lacking
␣
-synuclein. J Neurosci
22:8797– 8807.
Chandra S, Chen X, Rizo J, Jahn R, Sudhof TC (2003) A broken alpha-helix
in folded alpha-synuclein. J Biol Chem 278:15313–15318.
Chandra S, Fornai F, Kwon HB, Yazdani U, Atasoy D, Liu X, Hammer RE,
Battaglia G, German DC, Castillo PE, Sudhof TC (2004) Double-
knockout mice for alpha- and beta-synucleins: effect on synaptic func-
tions. Proc Natl Acad Sci USA 101:14966 –14971.
Chi P, Greengard P, Ryan TA (2001) Synapsin dispersion and reclustering
during synaptic activity. Nat Neurosci 4:1187–1193.
Chi P, Greengard P, Ryan TA (2003) Synaptic vesicle mobilization is regu-
lated by distinct synapsin I phosphorylation pathways at different fre-
quencies. Neuron 38:69 –78.
Cole NB, Murphy DD, Grider T, Rueter S, Brasaemle D, Nussbaum RL
(2002) Lipid droplet binding and oligomerization properties of the Par-
kinson’s disease protein alpha-synuclein. J Biol Chem 277:6344 –6352.
Davidson WS, Jonas A, Clayton DF, George JM (1998) Stabilization of
alpha-synuclein secondary structure upon binding to synthetic mem-
branes. J Biol Chem 273:9443–9449.
Ehlers MD (2003) Activity level controls postsynaptic composition and sig-
naling via the ubiquitin-proteasome system. Nat Neurosci 6:231–242.
Eliezer D, Kutluay E, Bussell Jr R, Browne G (2001) Conformational prop-
erties of alpha-synuclein in its free and lipid-associated states. J Mol Biol
307:1061–1073.
Elson EL, Qian H (1989) Interpretation of fluorescence correlation spec-
troscopy and photobleaching recovery in terms of molecular interactions.
Methods Cell Biol 30:307–332.
Fernandez-Alfonso T, Ryan TA (2004) The kinetics of synaptic vesicle pool
depletion at CNS synaptic terminals. Neuron 41:943–953.
Fortin DL, Troyer MD, Nakamura K, Kubo S, Anthony MD, Edwards RH
(2004) Lipid rafts mediate the synaptic localization of
␣
-synuclein.
J Neurosci 24:6715– 6723.
Galvin JE, Uryu K, Lee VM, Trojanowski JQ (1999) Axon pathology in Par-
kinson’s disease and Lewy body dementia hippocampus contains alpha-,
beta-, and gamma-synuclein. Proc Natl Acad Sci USA 96:13450 –13455.
Gandhi SP, Stevens CF (2003) Three modes of synaptic vesicular recycling
revealed by single-vesicle imaging. Nature 423:607– 613.
Goldberg MS, Pisani A, Haburcak M, Vortherms TA, Kitada T, Costa C, Tong
Y, Martella G, Tscherter A, Martins A, Bernardi G, Roth BL, Pothos EN,
Calabresi P, Shen J (2005) Nigrostriatal dopaminergic deficits and hy-
pokinesia caused by inactivation of the familial Parkinsonism-linked gene
DJ-1. Neuron 45:489 – 496.
Gorell JM, Rybicki BA, Johnson CC, Peterson EL (1999) Smoking and Par-
kinson’s disease: a dose-response relationship. Neurology 52:115–119.
Henkel AW, Simpson LL, Ridge RM, Betz WJ (1996) Synaptic vesicle move-
ments monitored by fluorescence recovery after photobleaching in nerve
terminals stained with FM1-43. J Neurosci 16:3960 –3967.
Higgins D, Banker GA (1998) Primary dissociated cell cultures. In: Cultur-
ing nerve cells, Ed 2 (Banker GA, Goslin K, eds) pp 37–78. Cambridge,
MA: MIT.
Iwai A, Masliah E, Yoshimoto M, Ge N, Flanagan L, de Silva HA, Kittel A,
Saitoh T (1995) The precursor protein of non-A beta component of
Alzheimer’s disease amyloid is a presynaptic protein of the central ner-
vous system. Neuron 14:467– 475.
Jakes R, Spillantini MG, Goedert M (1994) Identification of two distinct
synucleins from human brain. FEBS Lett 345:27–32.
Jensen PH, Nielsen MS, Jakes R, Dotti CG, Goedert M (1998) Binding of
alpha-synuclein to brain vesicles is abolished by familial Parkinson’s dis-
ease mutation. J Biol Chem 273:26292–26294.
Jo E, McLaurin J, Yip CM, St George-Hyslop P, Fraser PE (2000) Alpha-
synuclein membrane interactions and lipid specificity. J Biol Chem
275:34328 –34334.
Kraszewski K, Daniell L, Mundigl O, De Camilli P (1996) Mobility of syn-
aptic vesicles in nerve endings monitored by recovery from photobleach-
ing of synaptic vesicle-associated fluorescence. J Neurosci 16:5905–5913.
Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H,
Epplen JT, Schols L, Riess O (1998) Ala30Pro mutation in the gene en-
coding alpha-synuclein in Parkinson’s disease. Nat Genet 18:106 –108.
Kubo SI, Nemani VM, Chalkley RJ, Anthony MD, Hattori N, Mizuno Y,
Edwards RH, Fortin DL (2005) A combinatorial code for the interaction
of alpha-synuclein with membranes. J Biol Chem 280:31664 –31672.
Lee HJ, Choi C, Lee SJ (2002) Membrane-bound alpha-synuclein has a high
aggregation propensity and the ability to seed the aggregation of the cy-
tosolic form. J Biol Chem 277:671– 678.
Li Z, Murthy VN (2001) Visualizing postendocytic traffic of synaptic vesi-
cles at hippocampal synapses. Neuron 31:593– 605.
Lippincott-Schwartz J, Snapp E, Kenworthy A (2001) Studying protein dy-
namics in living cells. Nat Rev Mol Cell Biol 2:444 –456.
Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH (2003) Photobleach-
ing and photoactivation: following protein dynamics in living cells. Nat
Cell Biol [Suppl]:S7–S14.
Lowe AW, Madeddu L, Kelly RB (1988) Endocrine secretory granules and
neuronal synaptic vesicles have three integral membrane proteins in com-
mon. J Cell Biol 106:51–59.
Maroteaux L, Scheller RH (1991) The rat brain synucleins; family of pro-
teins transiently associated with neuronal membrane. Brain Res Mol
Brain Res 11:335–343.
Maroteaux L, Campanelli JT, Scheller RH (1988) Synuclein: a neuron-
10920 •J. Neurosci., November 23, 2005 •25(47):10913–10921 Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein

specific protein localized to the nucleus and presynaptic nerve terminal.
J Neurosci 8:2804 –2815.
McGehee DS, Heath MJ, Gelber S, Devay P, Role LW (1995) Nicotine en-
hancement of fast excitatory synaptic transmission in CNS by presynaptic
receptors. Science 269:1692–1696.
Murphy DD, Rueter SM, Trojanowski JQ, Lee VM (2000) Synucleins are
developmentally expressed, and
␣
-synuclein regulates the size of the pre-
synaptic vesicular pool in primary hippocampal neurons. J Neurosci
20:3214 –3220.
Narayanan V, Scarlata S (2001) Membrane binding and self-association of
alpha-synucleins. Biochemistry 40:9927–9934.
Outeiro TF, Lindquist S (2003) Yeast cells provide insight into alpha-
synuclein biology and pathobiology. Science 302:1772–1775.
Perrin RJ, Woods WS, Clayton DF, George JM (2000) Interaction of human
alpha-synuclein and Parkinson’s disease variants with phospholipids.
Structural analysis using site-directed mutagenesis. J Biol Chem
275:34393–34398.
Perrin RJ, Woods WS, Clayton DF, George JM (2001) Exposure to long
chain polyunsaturated fatty acids triggers rapid multimerization of
synucleins. J Biol Chem 276:41958 –41962.
Phair RD, Misteli T (2000) High mobility of proteins in the mammalian cell
nucleus. Nature 404:604 – 609.
Phair RD, Misteli T (2001) Kinetic modelling approaches to in vivo imag-
ing. Nat Rev Mol Cell Biol 2:898 –907.
Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B,
Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Atha-
nassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin
RC, Di Iorio G, Golbe LI, Nussbaum RL (1997) Mutation in the alpha-
synuclein gene identified in families with Parkinson’s disease. Science
276:2045–2047.
Quik M (2004) Smoking, nicotine and Parkinson’s disease. Trends Neuro-
sci 27:561–568.
Sankaranarayanan S, Ryan TA (2000) Real-time measurements of vesicle-
SNARE recycling in synapses of the central nervous system. Nat Cell Biol
2:197–204.
Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Huli-
han M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Han-
son M, Maraganore D, Adler C, Cookson MR, Muenter M, Baptista M,
Miller D, Blancato J, et al. (2003) Alpha-synuclein locus triplication
causes Parkinson’s disease. Science 302:841.
Snapp EL, Hegde RS, Francolini M, Lombardo F, Colombo S, Pedrazzini E,
Borgese N, Lippincott-Schwartz J (2003) Formation of stacked ER cis-
ternae by low affinity protein interactions. J Cell Biol 163:257–269.
Snyder H, Mensah K, Theisler C, Lee J, Matouschek A, Wolozin B (2003)
Aggregated and monomeric alpha-synuclein bind to the S6⬘proteasomal
protein and inhibit proteasomal function. J Biol Chem 278:11753–11759.
Specht CG, Tigaret CM, Rast GF, Thalhammer A, Rudhard Y, Schoepfer R
(2005) Subcellular localisation of recombinant alpha- and gamma-
synuclein. Mol Cell Neurosci 28:326 –334.
Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M (1998a)
Alpha-synuclein in filamentous inclusions of Lewy bodies from Parkin-
son’s disease and dementia with Lewy bodies. Proc Natl Acad Sci USA
95:6469 – 6473.
Spillantini MG, Crowther RA, Jakes R, Cairns NJ, Lantos PL, Goedert M
(1998b) Filamentous alpha-synuclein inclusions link multiple system at-
rophy with Parkinson’s disease and dementia with Lewy bodies. Neurosci
Lett 251:205–208.
Tanner CM, Goldman SM, Aston DA, Ottman R, Ellenberg J, Mayeux R,
Langston JW (2002) Smoking and Parkinson’s disease in twins. Neurol-
ogy 58:581–588.
Tofaris GK, Layfield R, Spillantini MG (2001) Alpha-synuclein metabolism
and aggregation is linked to ubiquitin-independent degradation by the
proteasome. FEBS Lett 509:22–26.
Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury Jr PT (1996)
NACP, a protein implicated in Alzheimer’s disease and learning, is na-
tively unfolded. Biochemistry 35:13709 –13715.
Withers GS, George JM, Banker GA, Clayton DF (1997) Delayed localiza-
tion of synelfin (synuclein, NACP) to presynaptic terminals in cultured
rat hippocampal neurons. Brain Res Dev Brain Res 99:87–94.
Yavich L, Tanila H, Vepsalainen S, Jakala P (2004) Role of
␣
-synuclein in
presynaptic dopamine recruitment. J Neurosci 24:11165–11170.
Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E, Ros R, Ampuero I, Vidal
L, Hoenicka J, Rodriguez O, Atares B, Llorens V, Gomez Tortosa E, del Ser
T, Munoz DG, de Yebenes JG (2004) The new mutation, E46K, of alpha-
synuclein causes Parkinson and Lewy body dementia. Ann Neurol
55:164 –173.
Zhou FM, Liang Y, Dani JA (2001) Endogenous nicotinic cholinergic activ-
ity regulates dopamine release in the striatum. Nat Neurosci 4:1224 –
1229.
Fortin et al. •Activity-Dependent Dynamics of
␣
-Synuclein J. Neurosci., November 23, 2005 •25(47):10913–10921 • 10921