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The Journal of Neuroscience, May 1, 2000, 20(9):3214-3220
Synucleins Are Developmentally Expressed, and  -Synuclein
Regulates the Size of the Presynaptic Vesicular Pool in Primary
Hippocampal Neurons
Diane D.
Murphy,
Susan M.
Rueter,
John Q.
Trojanowski, and
Virginia M.-Y.
Lee
Center for Neurodegenerative Disease Research, Department of
Pathology and Laboratory Medicine, University of Pennsylvania Medical
School, Philadelphia, Pennsylvania 19104
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ABSTRACT |
 -,  -, and  -Synuclein, a novel family of neuronal proteins,
has become the focus of research interest because -synuclein has
been increasingly implicated in the pathogenesis of Parkinson's and
Alzheimer's disease. However, the normal functions of the synucleins
are still unknown. For this reason, we characterized -, -, and
-synuclein expression in primary hippocampal neuronal cultures and
showed that the onset of - and -synuclein expression was delayed
after synaptic development, suggesting that these synucleins may not be
essential for synapse formation. In mature cultured primary neurons,
- and -synuclein colocalized almost exclusively with
synaptophysin in the presynaptic terminal, whereas little -synuclein
was expressed at all. To assess the function of -synuclein, we
suppressed expression of this protein with antisense oligonucleotide
technology. Morphometric ultrastructural analysis of the -synuclein
antisense oligonucleotide-treated cultures revealed a significant
reduction in the distal pool of synaptic vesicles. These data suggest
that one function of -synuclein may be to regulate the size of
distinct pools of synaptic vesicles in mature neurons.
Key words:
Lewy bodies; Parkinson's disease; primary neurons; synapse; synuclein; vesicle
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INTRODUCTION |
-Synuclein was first isolated in
1988 from synaptic vesicles of Torpedo californica and rat
brain (Maroteaux et al., 1988 ). Subsequently, other members of this
family of neuronal proteins have been identified: -synuclein, also
found mainly in the CNS, -synuclein, more abundant in the PNS than
in the CNS, and synoretin, present primarily in the retina (Nakajo et
al., 1990; Jakes et al., 1994; Ji et al., 1997; Buchman et al., 1998;
Surguchov et al., 1999). -Synuclein has been shown to be a primary
component of Lewy bodies, the neuropathological hallmarks of sporadic
Parkinson's disease (PD) (Spillantini et al., 1997; Baba et al.,
1998), and pathogenic -synuclein gene mutations have been identified
in familial forms of PD (Polymeropoulos et al., 1997; Kruger et al., 1998). Moreover, -synuclein has been detected in Lewy bodies that
are diagnostic of the Lewy body variant of Alzheimer's Disease (LBVAD)
as well as dementia with Lewy bodies (DLB) (Spillantini et al., 1997;
Baba et al., 1998; Trojanowski et al., 1998). Thus, insight into
mechanisms of synuclein pathology may be critical for understanding
brain degeneration in PD, LBVAD, and DLB (Trojanowski et al.,
1998).
The implication of -synuclein in neurodegenerative disease has
stimulated efforts to elucidate the normal distribution and functions
of -, -, and -synuclein. For example, because -synuclein is
localized to presynaptic terminals throughout the adult mammalian brain
and may appear earlier than synaptophysin during CNS development, it
has been suggested to play a role in synaptogenesis (Hsu et al., 1998).
However, in vitro studies show that the vesicle-associated protein synapsin I appears as early as 24 hr, whereas -synuclein is
not detected until day 5 (Withers et al., 1997). Thus, it is unclear
whether -synuclein is needed for the development of synapses or for
the maturation and modulation of previously existing ones. There is
also evidence that -synuclein may associate with synaptic vesicles
because -synuclein has been detected in vesicular fractions of human
brain (Irizarry et al., 1996) and binds to both synthetic membranes (Davidson et al., 1998) and vesicles isolated from rat brain
(Jensen et al., 1998).
The present study assessed -, -, and -synuclein expression in
primary hippocampal neurons grown at a substantially increased density.
These high-density cultures exhibit a robust, rapid development of
synaptic contacts and spines (Papa et al., 1995) that corresponds to
those seen during postnatal hippocampal development. Moreover, the
synaptic ultrastructure of high-density cultures resembles that found
in situ (Bartlett and Banker, 1984). We used light microscopic immunocytochemistry, electron microscopy, and protein biochemistry to characterize the expression of the synuclein family of
proteins in hippocampal neurons up to 3 weeks in culture. We also
examined changes in synaptic structure after treatment of the neurons
with antisense (AS) oligonucleotides to reduce -synuclein expression. Here, we report that - and -synuclein were expressed after synaptophysin and localized almost exclusively to presynaptic terminals of mature neurons. In addition, downregulation of
-synuclein by AS oligonucleotides caused a selective reduction in
the size of the presynaptic vesicular pool. Our data suggest that
-synuclein may interact with and regulate specific pools of synaptic
vesicles, thereby modulating synaptic functions in the normal brain.
These findings have important implications for the role -synuclein may play in neurodegenerative disease.
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MATERIALS AND METHODS |
Hippocampal cultures. High-density hippocampal
neuronal cultures were prepared as previously described (Papa et al.,
1995). Briefly, 20-d-old embryos were taken from anesthetized Sprague Dawley rats. The brains were removed and placed in ice-cold (4°C) L-15 medium supplemented with 0.6% glucose and 15 µg/ml gentamicin. The hippocampus was dissected and mechanically disaggregated by gentle
trituration using a Pasteur pipette. Dissociated cells (500,000 cells/well) were plated onto sterile 12 mm glass or Thermanox coverslips that were coated with collagen (50 µg/ml) and
poly-L-lysine (15 µg/ml). Cells were also prepared on
six-well tissue culture dishes for Western blot analysis. Thermanox
coverslips were used for electron microscopy (see below). The plating
medium was Eagle's MEM containing 5% heat-inactivated horse
serum, 5% fetal calf serum, 2 mM glutamine, 0.6% glucose,
and 15 µg/ml gentamicin. Cells were incubated at 37°C with 5%
CO2. The first change of medium, ~4-6 d after
plating, included 50 µg/ml uridine and 20 µg/ml deoxyuridine to
prevent glial cell overgrowth. The cultures were fed thereafter 1-2
times a week, with Eagle's MEM as above.
Immunocytochemistry. Cells were fixed in 4%
paraformaldehyde in PBS. After blocking and permeabilization in
5% goat serum with 0.1% saponin, cells were incubated with primary
antibodies to the following: Syn102 (recognizes and synuclein),
SNL-1 (-synuclein-specific), syn207 (-synuclein-specific) (Tu et
al., 1998), and a rabbit polyclonal antiserum specific for
-synuclein, VAMP (Chemicon, Temecula, CA), synaptophysin (Boehringer
Mannheim, Mannheim, Germany), syntaxin (Upstate Biotechnology, Lake
Placid, NY), synapsin I (Molecular Probes, Eugene, OR), and MAP2
(Upstate Biotechnology). The anti-synuclein antibodies used here were
previously characterized, and the specificity of each antibody for
-, -, and -synuclein was determined (Giasson et al., 2000).
Cells were washed and incubated with FITC- or rhodamine-conjugated
secondary antibodies. Cells were then imaged on a Leica (Nussloch,
Germany) confocal laser-scanning microscope, using a 1.4 NA,
100× oil immersion objective lens.
Western blot analysis. Total protein extracts were prepared
from 6-well dishes of primary hippocampal neurons in
detergent-containing cell extraction buffer (50 mM Tris
HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.1%
Triton X-100, 0.5 mM PMSF, and 1 mM DTT,
protease inhibitor cocktail). Harvested cells were spun at 100,000 × g in a TL100 ultracentrifuge (Beckman). Protein
concentration in the extracts was determined by the Bradford assay.
Approximately 30 µg of the supernatant was fractionated by
SDS-PAGE and transferred onto nitrocellulose. The blots were
incubated in blocking solution containing 5% nonfat dry milk in 1×
Tris-buffered saline (TBS) and then incubated overnight at 4°C with
the various primary antibodies described above diluted in 5% milk/TBS.
As a control, blots were also stained for neuron-specific enolase (NSE;
Polysciences, Warrington, PA) to normalize the amount of neuronal
protein. The blots were washed in TBS-T (TBS + 0.1% Tween 20) and
incubated for 2 hr at room temperature with secondary antibodies
[125I] conjugated to either anti-mouse
IgG or Protein A at a concentration of 1 µCi/ml of PTX buffer (10 mM sodium phosphate, pH 7.3, 1 mM EDTA, 150 mM NaCl, 0.2%
Triton X-100, and 4% BSA). The blots were washed again with TBS-T and
exposed in a PhosphorImager cassette (Molecular Dynamics, Sunnyvale,
CA) for 24-48 hr. The protein levels were quantified using ImageQuant
software (Molecular Dynamics), each lane normalized to NSE levels.
Antisense treatments. Oligonucleotides were prepared from
the 5' end of the coding region for rat -synuclein. The antisense (AS) oligonucleotide sequence was 5'-CCTTTCATGAACAC ATCCATGGC-3'. The
reverse sense (S) oligonucleotide sequence (5'-GCCATGGA
TGTGTTCATGAAAGG-3') and a scrambled oligonucleotide sequence
(5'-TAGCTCGCTACGTAATCACCACT-3') served as controls. All
oligonucleotides contained a phosphorothioate group at every residue.
Cells were placed in N3 serum-free media (Banker and Goslin, 1991) on
the day before and during AS oligonucleotide treatments to provide
better delivery of oligonucleotides to the cells. Cells [6 d in
vitro (div)] received 5 µM AS or S
oligonucleotide every other day for an additional 6 d. To assay
-synuclein involvement in synaptic development, an additional set of
cultures were dosed at 3 div and continuously for 2 weeks thereafter,
and subsequently stained for synaptophysin.
Electron microscopy. Control, AS and S
oligonucleotide-treated cells (four experiments in four separate sets
of cultures) grown on Thermanox coverslips were fixed in modified
Karnoversusky's fixative (Electron Microscopy Sciences) consisting of
3% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M
phosphate buffer. Cells were then dehydrated in graded series of
ethanol and embedded in araldite. Thin sections were cut on an
UltracutE, stained, and observed in a JEOL 100 microscope at 33,000×.
Synapses were selected using a modified protocol as previously
described (Reist et al., 1998). Briefly, only those synapses with a
well-defined postsynaptic density were included in the counting assay,
and grids were scanned so as not to photograph the same synapse twice.
Negatives were scanned and calibrated using NIH Image software.
Cross-sectional area of each synapse was measured, and the number of
vesicles counted as previously described (Pozzo-Miller et al., 1999).
Vesicles touching the synaptic membrane, or those within less than a
vesicle diameter to the membrane were counted as "docked." The
others were counted as the "distal" vesicular pool. Counts were
divided by the cross-sectional area of the synapse, and means and
SEs were calculated. Seventy-five, 97, and 56 synapses
were counted for the control, antisense, and sense treatment groups, respectively.
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RESULTS |
The expression of -, -, and -synuclein was monitored by
indirect immunofluorescence from 2-21 div. While synaptophysin was
observed within the Golgi complex and early synapses as soon as 3 div,
- and -synuclein were not expressed until 6 div. At this time,
-synuclein (Fig. 1a) and
-synuclein (data not shown) were detected primarily in the cytosol
throughout the cell body and primary processes, but also in a few
synapses. By 14 div, - and -synuclein were predominantly
localized to the presynaptic terminal, as evidenced by colocalization
with synaptophysin (a well characterized presynaptic protein), and at 3 weeks they appeared almost exclusively at the presynapse (Fig.
1b,c). At 3 weeks, these neurons are considered to be
"mature" because they demonstrate a stable number of excitatory
connections, dendritic outgrowth, and dendritic spine density that
increase no further during the culture life span (Papa et al., 1995).
The expression of - and -synuclein was not confined to a
particular type of synapse, excitatory or inhibitory, because they
colocalized with glutamic acid decarboxylase (GAD) which specifically
labels inhibitory GABAergic synapses (Fig. 1d). Both -
and -synuclein showed the same pattern of development and
colocalized with one another (Fig. 1e). Because parallel
studies revealed very few -synuclein-positive boutons at any time
point (Fig. 1f), -synuclein was not included in
further experiments.
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Figure 1.
Synuclein characterization by immunofluorescence.
1 (a)-, 2 (b)-, and 3 (c)-week-old cultures stained for -synuclein
(red) and synaptophysin (green).
Cytosolic -synuclein staining is evident in a.
d, Double labeling of -synuclein
(green) and GAD (red) show
colocalization in an inhibitory neuron and in GABAergic presynaptic
terminals (yellow). e, - and
-synuclein are colocalized (yellow) to the
presynaptic terminal. f, -synuclein
(red) is poorly expressed in mature hippocampal neurons
as compared to synaptophysin (green). The nuclei
in a-f are labeled by Hoechst
(blue) dye. Scale bar, 10 µm.
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Total - and -synuclein expression levels were also monitored by
quantitative Western analysis at 1, 2, and 3 weeks in culture. Western
analysis indicated that -synuclein expression peaked at 1 week and
decreased by ~25% at 2 weeks and ~40% at 3 weeks. -Synuclein
expression levels did not change significantly over the same time
period in culture (Fig. 2).
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Figure 2.
Synuclein protein expression in cultured
hippocampal neurons. - and -synuclein expression in neurons at 1, 2, 3 weeks in culture was determined by quantitative Western blot
analysis from Triton X-100 soluble cell extracts
(a). The blots were probed with antibody syn102
(specific for both - and -synucleins) and an antibody to NSE.
b, Quantitation of - and -synuclein expression at
1, 2, and 3 weeks in culture. Closed bars represent
-synuclein expression, and open bars represent
-synuclein expression. All synuclein signals were normalized to the
signal for NSE to account for the amount of neuronal protein loaded per
lane. Results are presented as the average percentage of
expression ± SEM. The expression at 1 week was arbitrarily set at
100%. n = 3; *p < 0.05.
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To probe the functional role of -synuclein in the presynaptic
terminal, antisense (AS) oligonucleotides were engineered and delivered
to the cells to prevent -synuclein expression. A sense (S)
oligonucleotide was used as a control as were cells that received no
oligonucleotide treatment and were grown in serum-free medium. Cells
treated for up to 6 div with AS oligonucleotide displayed -synuclein
immunofluorescence that was substantially reduced compared to control
or S oligonucleotide-treated cultures (Fig. 3), however, MAP2 staining was unchanged,
indicating that the AS oligonucleotide did not disrupt normal dendritic
development (data not shown). Quantitative Western blots showed that
-synuclein expression was decreased to 53 ± 3.1% of controls
by AS oligonucleotide treatment, whereas the levels of -synuclein
and NSE were unaffected (Fig.
4a,b). Treatment of the cells
with a sense or scrambled oligonucleotide had no effect on the level of
expression of -synuclein (Fig. 4a; data not shown).
Although - and -synuclein exhibit sequence homology, the
-synuclein-specific AS oligonucleotide had little effect on
-synuclein expression.
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Figure 3.
A specific AS oligonucleotide decreases
-synuclein expression. Cultured hippocampal neurons were
either not treated (a), or treated with AS
oligonucleotide (b) or with S oligonucleotide
(c) for 6 d and immunostained with antibody
SNL-1 (specific for -synuclein). Scale bar, 10 µm.
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Figure 4.
AS oligonucleotide treatment effectively decreases
-synuclein protein expression. a, Quantitative
Western blot analysis of -synuclein and NSE levels in control
(C), antisense (AS)-treated, and
sense (S)-treated cultures for 6 d.
b, Quantitation of - and -synuclein levels in
AS-treated cultures compared to control (untreated) cells. No
differences were found between control and sense
oligonucleotide-treated cultures. The closed bar
represents -synuclein expression, and the open bar
represents -synuclein expression. Results are presented as the
average percentage of expression of control cells ± SEM.
n = 3; *p < 0.05.
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Cultures were also assessed for other proteins found in the presynaptic
terminal to determine if they were affected by -synuclein suppression. The immunofluorescence localization of syntaxin, synaptophysin, synapsin I, and synaptobrevin, or vesicle-associated membrane protein (VAMP) showed little or no change after AS
oligonucleotide treatment (Fig. 5).
However, the staining intensity of vesicular boutons appeared to be
slightly decreased for synapsin I (Fig. 5, compare a, b) and
synaptophysin (Fig. 5, compare c, d). Examination of the
levels of these synaptic proteins by quantitative Western analysis
showed that synaptophysin and synapsin I indeed decreased in the AS
oligonucleotide-treated cells (68 ± 10.1% and 63 ± 9.7% of control, respectively), whereas VAMP, syntaxin, and NSE showed no
significant change (Fig. 6).
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Figure 5.
AS treatment results in an overall decrease of
several presynaptic proteins. Left panels (a, c,
e, g) are control cultures, right panels
(b, d, f, h) are AS oligonucleotide-treated cultures.
Synapsin I (a, b), synaptophysin (c, d),
VAMP (e, f), and syntaxin (g,
h). Although diminished fluorescent puncta were observed in
b and d, no changes in the localization
of these presynaptic proteins were detected. Scale bar, 10 µm.
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Figure 6.
Effect of -synuclein AS oligonucleotide
treatment on the expression of synaptic proteins. Quantitative Western
blot analysis of synaptophysin, VAMP, syntaxin, and synapsin I levels
in AS-treated cultures. All protein signals were normalized to the
signal for NSE to account for the amount of neuronal protein loaded per
lane. The open bar, dotted bar, gray bar, and
black bar represent synaptophysin (SP)
expression, VAMP expression, syntaxin expression, and synapsin I
expression, respectively. Results are presented as the average
percentage of expression of control cells ± SEM.
n = 3; *p < 0.05.
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To determine the ultrastructural consequences of -synuclein
depletion on the presynaptic terminal, we compared the morphological characteristics of synapses from control, AS, and S
oligonucleotide-treated cultures using electron microscopy. Random
synapses that had readily observable postsynaptic densities were
photographed at 33,000×, and the vesicles were counted in each
presynapse. No distinction was made between symmetrical or asymmetrical
synapses, because -synuclein is found in both excitatory and
inhibitory terminals, and discrimination of synapse type in cultured
neurons is somewhat unreliable. Samples were pooled from four different
cultures grown from different animals. Control values were comparable
to those described for primary hippocampal neurons (Harris and Sultan, 1995; Schikorski and Stevens, 1997; Boyer et al., 1998). While the
number of "docked" vesicles did not differ between the groups, the
"distal" synaptic vesicle pool size was significantly reduced in
the AS oligonucleotide-treated group as compared to the controls (Fig.
7). Statistical analysis of the vesicle
numbers per square micrometer of synaptic area indicated that AS
oligonucleotide-treated cultures had significantly fewer distal
vesicles, whereas the docked vesicles in this group were unchanged
(Table 1). The number of vesicles per
square micrometer of synaptic area in S oligonucleotide-treated cells
was somewhat higher than that in control cultures.
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Figure 7.
-Synuclein AS oligonucleotide treatment
decreases the synaptic vesicle pool at the presynapse. Left
panels (a, c, e) are representative images of
synapses from control hippocampal neurons. Right panels
(b, d, f) are images of representative synapses
from antisense oligonucleotide-treated hippocampal neurons. Note that
synapses from both groups have a similar number of docked vesicles and
possess well-defined postsynaptic densities. However, vesicles distal
to the docking region are depleted in the AS oligonucleotide-treated
cells (compare a, c, e with b, d,
f). Scale bar, 200 nm.
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DISCUSSION |
It is now widely recognized that -synuclein plays an important
role in the pathogenesis of several neurodegenerative diseases known
collectively as synucleinopathies (Trojanowski et al., 1998). For
example, -synuclein is found in Lewy bodies and neuronal fibrous
cytoplasmic inclusions, as well as in Lewy neurites and dystrophic
neuronal processes in brains of PD, DLB, and LBVAD patients
(Spillantini et al., 1997; Baba et al., 1998). Also, -synuclein is
likely the major component of glial and neuronal inclusions in multiple
system atrophy (MSA) and Hallervorden-Spatz disease (Tu et al.,
1998). The importance of -synuclein lesions in the pathogenesis of
synucleinopathies is supported by the recent observations that
-synuclein gene mutations are autosomally dominant in a subset of
familial PD pedigrees (Polymeropoulos et al., 1997; Kruger et al.,
1998). In addition, mutated -synuclein, as well as wild-type,
aggregates into filaments in vitro (Conway et al., 1998;
Crowther et al., 1998; El-Agnaf et al., 1998; Giasson et al., 1999).
Despite our knowledge of these synuclein pathologies, little is known
about the normal function of -synuclein in the mammalian brain, and
we addressed this issue in the studies described here.
Although - and -synuclein were expressed almost exclusively at
presynaptic terminals in mature primary neurons, both showed a delayed
onset of expression and localization as compared to other presynaptic
proteins. Our data are consistent with a previous study in which the
expression of -synuclein was delayed in comparison to that of
synapsin I in low-density cultures of rat hippocampal neurons (Withers
et al., 1998). However, this previous study showed a dramatic
increase in -synuclein expression in 30 div cultures when compared
with 5 div cultures, whereas we demonstrated here that the level of
-synuclein expression peaks at 1 week and declines thereafter. These
differences may be attributable to the fact that low-density cultures
often form autaptic connections and synaptic development may differ
from that of the high-density cultures used in our study. Here, it was
shown that - and -synuclein expression at the synapse followed
that of synaptophysin by at least a day. Whereas -synuclein levels
were higher at 1 week versus later time points and -synuclein levels
did not significantly change over the culture period, both isoforms
showed the same delayed localization to synapses and eventually became
exclusively expressed at fully mature presynaptic terminals. This would
suggest that these synucleins may function in the maintenance, rather than in the initial formation, of synapses. The lack of gross morphological deficits in the brains of mice lacking -synuclein further indicates that -synuclein is not essential for neuronal development and differentiation (Abeliovich et al., 2000).
To gain insight into the function of -synuclein, we used a specific
AS oligonucleotide to decrease -synuclein expression in high-density
primary hippocampal neurons that exhibit a robust development of well
characterized synapses (Papa et al., 1995). By using an AS
oligonucleotide in culture, we avoid some of the pitfalls experienced
with genetic knock-outs that frequently do not demonstrate overt
phenotypic changes (Janz and Sudhof, 1995). The AS oligonucleotide was
successful in that it decreased -synuclein levels by ~50%,
whereas -synuclein levels remained unchanged. Because MAP2 staining
was also unchanged, the AS oligonucleotide treatment did not adversely
affect dendritic outgrowth. Moreover, -synuclein AS oligonucleotide
treatment had the most dramatic effects when given repeatedly for
several days, suggesting that presynaptic -synuclein may be
long-lived. However, when -synuclein expression was blocked shortly
after neurons were plated, this did not prevent the formation of
synaptophysin-immunoreactive boutons (data not shown), again suggesting
a role for -synuclein in synaptic maintenance rather than in the
initiation of synapse formation.
If -synuclein is involved in the maintenance of fully functional
synapses then a prolonged reduction in -synuclein levels may cause
changes in presynaptic morphology. Indeed, we detected a decrease in
the staining intensity at individual presynaptic boutons for synapsin I
and synaptophysin which was accompanied by a decrease in their protein
expression on quantitative Western blots. However, not all synaptic
proteins were down-regulated. For example, the levels of synaptobrevin
(VAMP) and syntaxin did not decrease significantly. Since syntaxin is
associated with the plasma membrane whereas synapsin I and
synaptophysin are associated with synaptic vesicles (Sudhof, 1995),
there may be differential consequences of -synuclein reduction on
several structures at the synapses. However, the significance of the
lack of a reduction in VAMP, also a synaptic vesicle protein, is
unclear at this time. Analysis of the distribution and level of
expression of presynaptic proteins in the -synuclein knockout mice
does not show decreased expression of synaptophysin and synapsin I as
we saw in the AS oligonucleotide-treated hippocampal cultures, although
this was determined by immunostaining brain sections and therefore is
not quantitative (Abeliovich et al. 2000).
Significantly, our ultrastructural analysis of presynaptic morphology
and vesicles support a decrease in synaptic vesicles. Although the
number of vesicles docked at the synaptic plasma membrane was
unchanged, AS oligonucleotide-treated cultures displayed a marked
reduction in the number of vesicles present in the distal pool,
suggesting a possible role for -synuclein in the regulation of the
store of vesicles available for transmitter release. Our EM results are
not consistent with those from the -synuclein knock-out mice, which
do not show any difference in synaptic vesicles (Abeliovich et al.
2000). However, vesicles were not counted and a statistical analysis
was not performed in the knockout mice. Additionally, the differences
could be caused by the analysis of neurons from different brain regions
(hippocampus vs striatum) or the age of the neurons (embryonic vs adult).
Our results are consistent with a role for -synuclein in the
maintenance of previously existing mature synapses and the
stabilization of synaptic function. There is much evidence to suggest
that the distal pool of synaptic vesicles is anchored by the actin
cytoskeleton in the presynaptic terminal, and numerous studies have
demonstrated that this binding is mediated by synapsin I (for review,
see Greengard et al., 1994). Synapsin I, which is peripherally
associated with the vesicle membrane, can bind vesicles to actin in a
phosphorylation-dependent manner (Stefani et al., 1997), and
-synuclein may potentiate or stabilize this interaction.
-Synuclein has been shown to bind to artificial phospholipid
membranes (Davidson et al., 1998) as well as to synaptic vesicles
(Jensen et al., 1998), which would support a vesicle-anchoring
function. It is known that synapsin I is phosphorylated by CaMKII
(Benfenati et al., 1992) present on presynaptic vesicles and
-synuclein is also phosphorylated on serine residues by CaMKII
(Nakajo et al., 1993). Therefore, perhaps -synuclein is also
phosphorylated by kinases present at the presynaptic terminal.
Furthermore, a significant fraction of synapsin I is transported via
slow component b (Greengard et al., 1993), which may also be the means
of transport for -synuclein (Jensen et al., 1998). Thus,
similarities exist between synapsin I and the synucleins, suggesting
they may regulate vesicles in a similar manner. Alternatively,
-synuclein may modulate the expression, regulation, or activity of
synapsin I itself. Therefore, alterations in -synuclein would likely
affect the vesicular pool.
These observations have implications relevant to mechanisms of
-synuclein pathologies and their role in PD and AD because our data
suggest that -synuclein is a predominantly presynaptic protein
involved in the maintenance of synaptic vesicle pools in primary
neurons. It is known that the familial PD mutations in the
-synuclein gene can abolish the binding of -synuclein to
presynaptic vesicles (Jensen et al., 1998) and that -synuclein forms
Lewy bodies in sporadic PD. Thus, the data presented here may signify
that the availability of vesicles for release in familial and sporadic
PD brains could be compromised by -synuclein pathologies, thereby
leading to impaired synaptic function and degeneration.
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FOOTNOTES |
Received Nov. 11, 1999; revised Feb. 14, 2000; accepted Feb. 24, 2000.
This work was supported by grants from the National Institute on Aging.
We acknowledge Drs. N. B. Cole, R.W. Doms, and V. Zhukareva for
scientific contributions, Dr. R. Balice-Gordon for use of the confocal
microscope, and N. Shah and J. Sanzo of the electron microscopy
facility for EM preparations and use of the electron microscope.
D.M. and S.R. contributed equally to this work.
Correspondence should be addressed to Dr. Virginia M.-Y. Lee,
Department of Pathology and Laboratory Medicine, University of
Pennsylvania School of Medicine, Maloney 3, HUP, 3600 Spruce Street,
Philadelphia, PA 19104-4283. E-mail: vmylee{at}mail.med.upenn.edu.
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ABSTRACT
INTRODUCTION
