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The Journal of Neuroscience, May 15, 2000, 20(10):3736-3744
Synapsin III: Developmental Expression, Subcellular Localization,
and Role in Axon Formation
Adriana
Ferreira1, 2,
Hung-Teh
Kao3, 4,
Jian
Feng3,
Mark
Rapoport1, and
Paul
Greengard3
1 Department of Cell and Molecular Biology and
2 Institute for Neuroscience, Northwestern University,
Chicago, Illinois 60611, 3 Laboratory of Molecular and
Cellular Neuroscience, The Rockefeller University, New York, New York
10021, and 4 Department of Psychiatry, New York University
Medical Center, New York, New York 10016
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ABSTRACT |
We have investigated the developmental expression and subcellular
localization of synapsin III, the newest member of the synapsin family,
in cultured mouse hippocampal neurons. Our results indicate that
synapsin III is expressed early during development, with levels peaking
7 d after plating and declining thereafter. Synapsin III is highly
concentrated in growth cones. Using specific antisense oligonucleotides, we have also examined the effect of depleting synapsin III on neurite elongation and synaptogenesis. When synapsin III was suppressed immediately after plating, hippocampal neurons extended minor processes but failed to differentiate one of them as the
axon. The suppression of synapsin III after axonal elongation did not
affect the time course of synapse formation. The results indicate that
synapsin III has a developmental time course, a subcellular
localization, and a developmental function very different from those of
synapsin I and synapsin II.
Key words:
synapsin III; neuronal polarity; growth cones; axonal
elongation; synaptogenesis; antisense oligonucleotides
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INTRODUCTION |
The synapsins are a family of
neuron-specific phosphoproteins. Two members of this family, synapsin I
and synapsin II, were identified almost two decades ago. Distinct genes
encode these two synapsins, and their transcripts are alternatively
spliced and give rise to two isoforms (Südhof et al.,
1989 ). Both synapsins are highly concentrated at presynaptic nerve
terminals in central neurons, are associated with the cytoplasmic
surface of synaptic vesicles (DeCamilli et al., 1983, 1988; Finger et
al., 1990; Greengard et al., 1993), and play a role in synapse
formation. The injection of synapsins into Xenopus
blastomeres accelerates synapse formation (Lu et al., 1992; Schaeffer
et al., 1994; Valtorta et al., 1995). In addition, the suppression of
either synapsin I or synapsin II results in the inhibition of
synaptogenesis in hippocampal neurons (Chin et al., 1995; Ferreira et
al., 1995, 1996, 1998). Several studies have suggested distinct roles
for synapsin I and synapsin II during neuronal development. In cultured
hippocampal neurons, synapsin I plays a major role in axonal elongation
and branching (Chin et al., 1995), whereas synapsin II plays a major role in the initial elongation of undifferentiated processes (Ferreira et al., 1994, 1998).
Synapsin III, the newest member of the synapsin family, was identified
recently. Synapsin III is present in human, mouse, rat (Hosaka and
Südhof, 1998; Kao et al., 1998), and Xenopus (Kao et al., 1999) brain and is predominantly expressed in neurons. Analysis of the primary structure of synapsin III indicates that the
most conserved domains are A, C, and E, when compared with either
synapsin I or synapsin II. In addition, synapsin III, like the other
synapsins, is a peripheral membrane protein associated with synaptic
vesicles (Kao et al., 1998). Whether synapsin III plays a role similar
to those described for synapsin I and synapsin II during neuronal
development was the subject of the present study. We analyzed the
developmental expression and subcellular distribution of synapsin III
in cultured mouse hippocampal neurons and its role in neurite
elongation and synapse formation.
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MATERIALS AND METHODS |
Preparation of hippocampal cultures. Neuronal
cultures were prepared from the hippocampi of embryonic day 16 mice as
described previously (Goslin and Banker, 1991; Chin et al.,
1995). Briefly, embryos were removed, and their hippocampi were
dissected and freed of meninges. The cells were dissociated by
trypsinization (0.25% for 15 min at 37°C) followed by trituration
with a fire-polished Pasteur pipette and plated onto
poly-L-lysine-coated coverslips in MEM with 10% horse
serum. After 4 hr, the coverslips were transferred to dishes containing
an astroglial monolayer and maintained in MEM containing N2 supplements
(Bottenstein and Sato, 1979) plus ovalbumin (0.1%) and sodium pyruvate
(0.1 mM). For antisense experiments, the coverslips were
transferred, 2 hr after plating, to 35 mm dishes and incubated in
glia-conditioned MEM containing N2 supplements. Sense or antisense
oligonucleotides were added directly to the culture medium as described below.
Immunocytochemical procedures. Cultures were fixed for 20 min with 4% paraformaldehyde in PBS containing 0.12 M
sucrose. They were then permeabilized in 0.3% Triton X-100 in PBS for
4 min and rinsed twice in PBS. The cells were preincubated in 10% BSA in PBS for 1 hr at 37°C and exposed to the primary antibodies (diluted in 1% BSA in PBS) overnight at 4°C. Finally, the cultures were rinsed in PBS and incubated with secondary antibodies for 1 hr at
37°C. The following antibodies were used: anti-synapsin III
[antiserum RU316 (Kao et al., 1998); antiserum RU486],
anti- -tubulin (clone DM1A) and polyclonal anti-tubulin (Sigma, St.
Louis, MO), anti-synaptophysin (clone SY38; Boehringer Mannheim,
Indianapolis, IN), anti-synapsin I [clone 18.1 (Südhof et al.,
1989)], anti-synapsin II (clone 19.21), goat anti-mouse IgG
fluorescein-conjugated, and goat anti-rabbit IgG rhodamine-conjugated
(Boehringer Mannheim). In some experiments, cells exposed to tubulin
antibodies were incubated with biotin-conjugated rabbit anti-mouse IgG
(Sigma) for 1 hr at room temperature. The coverslips were then washed in PBS and incubated in mouse ExtrAvidin (Sigma) for 1 hr at
room temperature. Finally, the coverslips were washed in PBS and
incubated in a substrate solution containing 0.05%
3,3'-diaminobenzidine tetrahydrochloride and 0.075%
H2O2 (v/v) in 50 mM Tris, pH 7.6. The reaction was stopped after sufficient
color had developed by immersing the coverslips in deionized water. To
visualize growth cones, cells stained with tubulin antibody were
counterstained using either rhodamine- or biotin-conjugated phalloidin
(Molecular Probes, Eugene, OR).
The specificity of staining was verified by preabsorption of synapsin
III antibody (RU316) with an excess of peptide 1322 for 10 hr at 4°C.
Peptide 1322 corresponds to residues 512-530 within domain J of
synapsin III and was used to generate this antibody. Control
incubations in which this antibody was preabsorbed with BSA were also
performed, showing no change in specific immunoreactivity.
Pictures were taken using TMAX 400 ASA film on a Nikon
microscope equipped with a photographic camera. Films were scanned using a Polaroid Sprint SCAN 35 scanner. The acquired digital image
files were transferred to a Macintosh G4 Power personal computer, and
images were processed using Adobe Photoshop (Adobe Systems, Mountain
View, CA) and printed using a Tektronic Phaser II SDX printer.
Antisense oligonucleotides. The initial experiments were
performed using the antisense oligonucleotide AS-SIII3 (position  32
to 13; 5'-TGGGAATTCTCCTTCCACAC-3') located entirely within the
5'-untranslated region. The experiments were repeated using a
nonoverlapping oligonucleotide designated AS-SIII9 (position +4 to +23;
5'-AGCCGCCTCCGGAGGAAGTT-3'). Position 1 refers to the first nucleotide
of the coding sequence of mouse synapsin III (Kao et al., 1999). Both
antisense oligonucleotides (Oligos etc., Wilsonville, OR) were
S-modified in the last three bases in the 3'-terminal region. For
studies before axonogenesis, oligonucleotides were added at a
concentration of 50 µM every 24 hr to hippocampal neurons
that had been maintained in culture for 4 hr. For studies before
synaptogenesis, oligonucleotides were added at a concentration of 50 µM every 24 hr to hippocampal neurons that had been
maintained in culture for 4 d. Control cultures were treated with
the same concentration of the corresponding sense strand oligonucleotide.
Morphometric analysis. Control and sense- and
antisense-treated neurons were fixed at different intervals after
plating and stained with tubulin or biotin-conjugated phalloidin (as
described above). Cells stained using the tubulin antibody were used to determine total length and the length of minor processes. Cells stained
using biotin-conjugated phalloidin were used to determine the growth
cone area. Immunoreac- tive processes and growth cones from
randomly selected cells were then drawn using a camera lucida, and
total neurite length and growth cone area were measured using a
digitizing tablet. The proximal limit of the growth cone was defined as
the distal part of the neurite where the diameter is twice as big as
the neurite itself (Bradke and Dotti, 1997).
All results of the morphometric analysis were expressed as means ± SEM. Comparisons of the means between groups were made using a
paired t test (sense- or antisense-treated vs control samples) and one-way ANOVA followed by a Scheffe test for
multiple comparison of means.
Detection of synapses. Synapse formation was determined
using synaptophysin and synapsin I as synaptic markers. Images from randomly selected sense- and antisense-treated neurons were acquired at
40×, printed together at the same magnification, and then coded and
randomized for blind analysis. The number of synaptophysin- or synapsin
I-immunoreactive dots (presynaptic specializations) was determined
manually in 150 cells for each experimental condition.
Protein determination, electrophoresis, and immunoblotting.
Cultured cells were rinsed twice in warmed PBS, scraped into Laemmli buffer, and homogenized in a boiling water bath for 5 min. After centrifugation, supernatants were removed and stored at 80°C until
use. Protein concentration was determined by the method of Lowry et al.
(1951) as modified by Bensadoun and Weinstein (1976).
SDS-polyacrylamide gels were run according to Laemmli (1970).
Transfer of protein to Immobilon membranes (Millipore, Bedford, MA) and
immunodetection were performed according to the method of Towbin et al.
(1979) as modified by Ferreira et al. (1989). The following antibodies
were used: anti--tubulin (clone DM1A; Sigma), anti-synapsin I (clone
18.1), anti-synapsin II (clone 19.21), and anti-synapsin III (antiserum
RU316 and RU486). Secondary HRP-conjugated antibodies (Promega,
Madison, WI) followed by enhanced chemiluminescence reagents (Amersham,
Arlington Heights, IL) were used. X-ray films were exposed to
the immunoblots and analyzed using a Bio-Rad 700 flatbed scanner
(Bio-Rad, Hercules, CA) and Molecular Analyst software (Bio-Rad). Films
were scanned at 600 dpi using light transmittance, and volume analysis
was performed on the appropriate bands. The lower band was included in
the quantification of synapsin IIIa. Films were analyzed after
different exposure times to ensure the accuracy of quantitation.
Densitometric values were normalized using -tubulin present in the extracts.
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RESULTS |
Developmental expression of synapsin III in cultured
hippocampal neurons
Immunoblots were prepared from cells kept in culture for
various intervals and reacted with specific anti-synapsin III
antibodies (RU316 and RU486). The two antibodies tested gave
virtually identical results and recognized a major band at 63 kDa
corresponding to synapsin IIIa (Kao et al., 1998). Levels of synapsin
III increased with development in culture, reached their peak at 7 d after plating, and decreased thereafter (Fig.
1). The pattern of expression of synapsin
III contrasted with those of synapsin I and synapsin II. Both of these
proteins continued to increase in amount as the cells developed in
culture (Fig. 1).
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Figure 1.
Temporal pattern of expression of the synapsins in
cultured hippocampal neurons. Whole-cell extracts were prepared from
E16 mouse dissociated hippocampal neurons kept in culture from 3 hr to
30 d. Right, The proteins (30 µg of total
protein/lane) were separated by SDS-PAGE, and immunoblots were reacted
with antibodies specific for synapsin I (clone 18.1), synapsin II
(clone 19.21), or synapsin III (antiserum RU316). Left,
Densitometry of immunoreactive bands was performed, and the results
were normalized using tubulin as a control. Values represent means ± SEM for three experiments. The results are expressed as the percent
of the highest level detected.
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Localization of synapsin III in cultured hippocampal neurons
When placed in culture hippocampal neurons extend and
differentiate axons and dendrites following a sequence of well defined morphological changes (Dotti et al., 1988). Hippocampal neurons, initially round and surrounded by lamellipodial veils (stage I), extend
undifferentiated minor processes as soon as 4 hr after plating (stage
II). One day after plating the cells extend an axon and several short
minor processes (stage III) that will differentiate into dendrites
4 d later (stage IV). Synaptic contacts are established 5-7 d
after plating (Fletcher et al., 1991). Synapsin III was detected in
hippocampal neurons from the beginning of their morphological differentiation. In stage I (data not shown) and stage II and stage III
(Fig. 2) cells, synapsin III was
concentrated in all cell bodies and in ~75% of growth cones. In
stage III cells, very faint immunoreactivity for synapsin III was also
detected along the axon. In mature hippocampal neurons (stage IV-V
cells), synapsin III immunoreactivity could be detected as fine puncta
along the axon (Fig. 2). Occasionally, large immunoreactive axonal
spots were detected.
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Figure 2.
Localization of synapsin III in developing
hippocampal neurons. Hippocampal neurons were fixed 8 hr (stage II;
A, B, low magnification; C,
D, high magnification), 1 d (stage III; E,
F), and 7 d (stage IV; G, H)
after plating and double stained using tubulin (A, C, E,
G) and synapsin III (antiserum RU316; B, D, F,
H) antibodies. Synapsin III immunoreactivity was
concentrated in cell bodies and growth cones (gc;
arrowheads) in stage II and stage III cells. In stage IV
cells (H), a fine puncta of
immunoreactive material was detected along the processes.
ax, Axon. Scale bars: A, B, 20 µm; C, D, 20 µm; E, F, 20 µm;
G, H, 20 µm.
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Double-staining experiments were performed to determine whether
synapsin III was present at synaptic contacts. Synaptophysin, synapsin
I, and synapsin II were used as synaptic markers. In cultured
hippocampal neurons, the localization of these proteins at synapses has
been confirmed at the ultrastructural level (Fletcher et al., 1991;
Ferreira et al., 1995). Although some synapsin III-immunoreactive spots
colocalized with synaptophysin, synapsin I, or synapsin II, most of the
immunoreactivity for synapsin III was localized at extrasynaptic sites
(Fig. 3).
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Figure 3.
Comparison of the localization of synapsin III
with that of synaptic markers in cultured hippocampal neurons. After
7 d in culture, hippocampal neurons were fixed and double stained
with synapsin III (A, C, E) and synaptophysin
(B), synapsin I (D), or
synapsin II (F) antibodies. Synapsin III
immunoreactivity failed to colocalize at synaptic sites with any of the
synaptic markers used. Arrows point to synaptic sites
(B, D, F) devoid of synapsin III (A, C,
E). Arrowheads point to extrasynaptic sites rich
in synapsin III (A, C, E) and devoid of synaptophysin
(B), synapsin I (D), or
synapsin II (F). Scale bar, 10 µm.
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Phenotype of synapsin III-depleted hippocampal neurons
The localization of synapsin III to growth cones suggested the
possibility of a role for synapsin III in axonal elongation and/or
synapse formation. To evaluate this possibility, we suppressed synapsin
III expression by culturing hippocampal neurons in the presence of
antisense oligonucleotides. Two nonoverlapping 20 mer oligonucleotides
were designed based on the mouse synapsin III sequence (Kao et al.,
1999) and added at a final concentration of 50 µM. To
suppress the expression of synapsin III before axonal elongation,
oligonucleotides were added to 4-hr-old cultures, and cells were
analyzed 24 and 48 hr later. Both antisense oligonucleotides (designated AS-SIII3 and AS-SIII9) reduced the levels of synapsin III
by 75-80% when compared with sense-treated or nontreated control cells at 24 hr (Fig.
4A) and 48 hr (data not
shown).
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Figure 4.
Effect of synapsin III antisense oligonucleotides
on cultured hippocampal neurons. A, Western blot
analysis of synapsin III and tubulin expression in control
(C) and sense (S)- and
antisense (AS)-treated neurons kept in culture for 24 hr. B, C, Graphs showing the stage of development
(B) and the total length and length of minor
processes (C) of control and sense- and
antisense-treated hippocampal neurons kept in culture for 2 d.
Cells stained with tubulin using the DAB method were used for this
morphometric analysis. For quantification purposes cells were
considered to be in stage 3 when one of the processes was at least 20 µm longer than the rest of the processes. Values represent
means ± SEM. A total of 90 cells from three different experiments were
analyzed for each experimental condition. *, Differs from control or
sense-treated cells (p < 0.001; ANOVA).
MP, Minor processes; SIII, synapsin
III.
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Synapsin III-depleted hippocampal neurons displayed an abnormal
morphology during their initial phases of development (Figs. 4,
5). No differences were detected in the
number, morphology, and length of minor processes as compared with
sense-treated or nontreated control cells. In contrast, axonal
differentiation was impaired (Fig. 4C). More than 80% of
the synapsin III-depleted hippocampal neurons failed to elongate their
axons 24 or 48 hr after plating (Figs. 4B, 5). The
lack of axonal differentiation in synapsin III-depleted neurons
resulted in a significant decrease in total neurite length when
compared with untreated controls or sense-treated cells (Fig.
4C). The impaired axonal elongation observed in synapsin
III-depleted cells was accompanied by changes in the growth cones. A
significant increase in the size of the growth cones was detected in
synapsin III-depleted neurons 24 hr (46% increase) and 48 hr (51%
increase) after plating when compared with controls (Fig.
6).
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Figure 5.
Phenotype of synapsin III-depleted hippocampal
neurons. After 2 d in culture, sense (A, B)- and
antisense (C, D)-treated cells were fixed and double
stained with tubulin (A, C) and synapsin III (B,
D) antibodies. Synapsin III immunoreactivity was detectable
along the axons (ax) and was highly concentrated in cell
bodies and growth cones (gc) in sense-treated
neurons. Note the absence of synapsin III immunoreactivity in synapsin
III antisense-treated neurons. Scale bar, 20 µm.
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Figure 6.
Suppression of synapsin III resulted in a changed
appearance of growth cones. A, Graph showing the area of
growth cones in control and sense (S)- and
antisense (AS)-treated hippocampal neurons kept in
culture for 1 and 2 d. Values represent the means ± SEM. A
total of 90 growth cones from three different experiments were analyzed
for each experimental condition. *, Differs from control or
sense-treated cells (p < 0.01; ANOVA).
SIII, Synapsin III. B-G, Representative
growth cones from control (B, C) and sense (D,
E)- and antisense (F, G)-treated neurons. The
cells were fixed, stained with a tubulin antibody (B, D,
F), and counterstained using rhodamine-phalloidin
(C, E, G). Scale bar, 10 µm.
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To determine whether synapse formation occurs in the absence of
synapsin III, cells were allowed to grow for 4 d and then were
treated with 50 µM sense or antisense synapsin III
oligonucleotides every 24 hr. Either antisense synapsin III
oligonucleotide (AS-SIII3 and AS-SIII9) reduced the amount of synapsin
III by ~80% as determined by Western blot analysis. The presence of
synaptic contacts in control and sense- and antisense-treated neurons
was determined, 24 and 72 hr after the addition of the antisense
oligonucleotide, using synaptophysin as a synaptic marker (Fig.
7). Synapses were detected as early as 5 d after plating in both sense- and
antisense-treated cultures. Thus, the time course of synapse formation
was not altered in synapsin III-depleted neurons when compared with
sense-treated or nontreated controls. We also determined the number of
synapses in sense- and antisense-treated cultures double stained with
tubulin and synaptophysin antibodies. No significant differences in the number of synapses were detected after 7 d in culture in synapsin III-depleted neurons when compared with sense-treated controls (30 ± 5 vs 37 ± 4 synapses/cell, respectively; n = 150 cells from three independent experiments). Similar results were
obtained when synapses were counted in sense- and antisense-treated
cultures double stained with tubulin and synapsin I antibodies (42 ± 7 vs 50 ± 4 synapses/cell, respectively; n = 150 cells from three independent experiments).
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Figure 7.
Synapse formation in synapsin III-depleted
hippocampal neurons. After 4 d in culture, neurons were treated
for 72 hr with sense (A-D) or antisense
(E-H) oligonucleotides and analyzed by double
staining with tubulin (A, E) and synapsin III (B,
F) antibodies or tubulin (C, G) and
synaptophysin (D, H) antibodies. Note the
presence of synapses in both sense (D)- and
antisense (H)-treated neurons. Scale bars:
A, B, 20 µm; C, D, 20 µm; E,
F, 20 µm; G, H, 20 µm.
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Finally, we considered the possibility that the effect of synapsin III
depletion might be attributable in part to a decrease in the expression
of the other known members of the synapsin family. No significant
changes in synapsin I (89 ± 8%) or synapsin II (92 ± 10%)
levels were detected in antisense synapsin III-treated neurons when
compared with control cells.
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DISCUSSION |
The present findings provide strong evidence of a distinct role
for synapsin III, the most recently discovered member of the synapsin
family, in the establishment of neuronal polarity in central neurons.
The predominant expression of synapsin III during the early stages of
development, as well as its subcellular localization at extrasynaptic
sites, distinguishes synapsin III from the other members of the
synapsin family. Although the levels of synapsin I and synapsin II
correlate with the extent of synapse formation both in situ
and in culture (Lohmann et al., 1978; Goelz et al., 1981) (this
study), the highest levels of synapsin III are detected during the
first week in culture, a period that corresponds to active process
elongation. Consistent with a role in neurite outgrowth, synapsin III
is highly enriched in growth cones in cultured hippocampal neurons.
This localization contrasts with that of synapsin I and synapsin II,
which are detected in minor processes and the distal third of the axon
in developing neurons. The preferential localization of synapsin III in
growth cones prompted us to perform experiments to determine the role
of this protein during neurite elongation and the establishment of
polarity in cultured hippocampal neurons. We used primary hippocampal
cultures because (1) they represent a relatively homogeneous
population of neurons and (2) they differentiate in a reproducible
manner via a series of well characterized morphological changes (Dotti
et al., 1988). Immediately after dissociation, neurons are round and
partially or totally surrounded by lamellipodial veils (stage I). The
consolidation of these veils into short and undifferentiated processes
(stage II) takes place as early as 4 hr after plating. These processes
show no net elongation for almost a day, at which time one of them
begins to elongate at a rapid rate and becomes the axon (stage III). We
have shown previously that this sequence of events is altered by the
suppression of either synapsin I or synapsin II. Synapsin II
suppression impaired the normal elongation of minor processes and,
therefore, the transition between stage I and stage II (Ferreira et
al., 1994, 1998). On the other hand, the suppression of synapsin I
resulted in well differentiated axons, albeit shorter and less branched
ones than those observed in wild-type cells (Chin et al., 1995). Our
results indicate that hippocampal neurons depleted of synapsin III are capable of extending normal minor processes but fail to elongate and
differentiate their axons (transition between stage II and stage III).
This impairment in axonal differentiation is accompanied by a
significant enlargement of the growth cones. It has been suggested that
the differentiation of one minor process into an axon is preceded by
the enlargement of its growth cone (Bradke and Dotti, 1997). The
enlargement of a growth cone presumably results from an increase in
membrane addition because of a selected trafficking of organelles
toward the tip of the process that will elongate as an axon. The
impairment of axonal elongation by synapsin III suppression in a cell
in which the rate of trafficking of material to growth cones is not
affected could result in the abnormal accumulation of material and,
hence, in an increase in the size of the growth cones.
Changes not only in the size of the growth cone but also in the
dynamics of its cytoskeleton precede axonal differentiation (Bradke and
Dotti, 1999). It has been suggested that the local instability of actin
filaments in a given growth cone could act as a physiological signal
triggering axonal differentiation (Bradke and Dotti, 1999). We could
speculate that the lack of synapsin III in growth cones alters actin
dynamics, preventing axonal elongation. Other members of the synapsin
family have been implicated in the polymerization and bundling of actin
filaments (Baines and Bennett, 1986; Petrucci and Morrow, 1991;
Chilcote et al., 1994). Although no direct evidence is available
regarding the interaction of synapsin III with actin, its high degree
of homology (Kao et al., 1998) with the actin-binding domain of
synapsin I (Bähler et al., 1989) suggests that synapsin
III also plays a role in the organization of the actin cytoskeleton.
The suppression of synapsin III after axonal elongation but before
synaptogenesis did not alter the time course of synapse formation in
cultured hippocampal neurons. The lack of a prominent role for synapsin
III in synapse formation is consistent with its paucity at synaptic
sites. Synapsin III is present at much lower levels in nerve terminals
than are synapsin I and synapsin II, which are required for synapse
formation in cultured hippocampal neurons. The suppression of synapsin
I or synapsin II by either antisense oligonucleotides or homologous
recombinant techniques results in a delay in synapse formation (Chin et
al., 1995; Ferreira et al., 1995, 1996; Li et al., 1995; Rosahl et al.,
1995; Takei et al., 1995). Although the present results argue against
an important role for synapsin III in synapse formation, its
involvement in synaptogenesis cannot be excluded because biochemical
studies indicate that synapsin III is associated with synaptic
vesicles, although at a much lower level than are synapsins I and II
(Kao et al., 1998).
The data presented here indicate that synapsin III has a role during
neuronal development distinct from those of synapsins I and II. Further
analyses should provide additional insights into the participation of
synapsin III in the mechanisms underlying axonal differentiation in
central neurons.
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FOOTNOTES |
Received Sept. 16, 1999; revised Feb. 22, 2000; accepted March 2, 2000.
This work was supported by the National Alliance for Research on
Schizophrenia and Depression Young Investigator Award and Northwestern
Institute for Neuroscience start-up funds to A.F. and by National
Institutes of Health Grants MH 39327 and AG 15072 to P.G.
Correspondence should be addressed to Dr. Adriana Ferreira,
Northwestern Institute for Neuroscience, Searle Building Room 5-474, 320 East Superior Street, Chicago, IL 60611. E-mail:
a-ferreira{at}nwu.edu.
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TOP
ABSTRACT
INTRODUCTION
