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The Journal of Neuroscience, January 1, 1998, 18(1):93-103
Amphiphysin I Antisense Oligonucleotides Inhibit Neurite
Outgrowth in Cultured Hippocampal Neurons
Olaf
Mundigl1,
Gian-Carlo
Ochoa1,
Carol
David1,
Vladimir I.
Slepnev1,
Alexander
Kabanov2, and
Pietro
De
Camilli1
1 Department of Cell Biology and Howard Hughes Medical
Institute, Yale University School of Medicine, Boyer Center for
Molecular Medicine, New Haven, Connecticut 06510, and
2 Department of Pharmaceutical Sciences, University of
Nebraska Medical Center, Omaha, Nebraska 68198
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ABSTRACT |
Amphiphysin I is an SH3 domain-containing neuronal protein,
enriched in axon terminals, which was reported to act as a
physiological binding partner for dynamin I in synaptic vesicle
endocytosis. Rvs167 and Rvs161, the yeast homologs of amphiphysin I,
have been implicated in endocytosis, actin function, and cell polarity. Now we have explored the possibility that amphiphysin I also may have a
role in actin dynamics and cell polarity by testing the effect of
amphiphysin I suppression on neurite outgrowth. Freshly plated
hippocampal neurons were exposed to antisense oligonucleotides via a
new delivery system based on a polycationic amphipathic polymer, PS980.
Western blot analysis revealed that amphiphysin I levels steadily
increased with neuronal differentiation, whereas in antisense-treated
cultures amphiphysin I levels were reduced to ~10% of control levels
at 48 hr. Concomitantly, a collapse of growth cones and a severe
inhibition of neurite outgrowth and axon formation were observed. A
similar effect was observed previously after dynamin I suppression in
the same culture system (Torre et al., 1994 ). We also have found that
amphiphysin I and dynamin I colocalize in developing neurons at all
developmental stages and that a pool of both proteins is colocalized
with actin patches at the leading edge of growth cones. Our findings
suggest a conserved role of the amphiphysin protein family in the
dynamics of the cortical cell cytoskeleton and provide new evidence for
a close functional link between amphiphysin I and dynamin I.
Key words:
Rvs; endocytosis; synaptic vesicles; actin; dynamin; synaptojanin; growth cones
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INTRODUCTION |
Amphiphysin I is a neuronal
cytosolic protein concentrated in nerve terminals, which was identified
independently as an antigen enriched in chicken synaptic membranes
(Lichte et al., 1992), and as the dominant autoantigen in a rare human
condition, paraneoplastic stiff-man syndrome (De Camilli et al., 1993;
Folli et al., 1993; David et al., 1994). It comprises an NH2 terminal
region, which has the potential to form coiled-coil structures (David
et al., 1994; Sivadon et al., 1995), a central region, which is
evolutionarily poorly conserved (Lichte et al., 1992; David et al.,
1994), and a COOH-terminal SH3 domain (David et al., 1994). It is a
member of a protein family that includes at least another member in
higher vertebrates, referred to as amphiphysin II (BIN1, SH3p9)
(Sakamuro et al., 1996; Sparks et al., 1996; Butler et al., 1997;
Leprince et al., 1997), and two members in yeast, the Rvs167 and Rvs161 proteins (Crouzet et al., 1991; David et al., 1994; Sivadon et al.,
1995).
Recent studies have suggested that amphiphysin I is a physiological
partner of dynamin I, a GTPase that plays a key role in the endocytotic
reaction of synaptic vesicle membranes (for review, see De Camilli and
Takei, 1996). Dynamin I binds the SH3 domain of amphiphysin I via its
proline-rich COOH-terminal domain (David et al., 1996; Grabs et al.,
1997). Temperature-sensitive mutations of the dynamin I gene in
Drosophila (shibire gene) produce a powerful temperature-sensitive block in synaptic vesicle recycling at the stage
of deeply invaginated plasmalemmal pit (Koenig and Ikeda, 1989).
Dynamin oligomerizes into ring-like structures at the neck of
endocytotic pits, and the nonhydrolyzable analog of GTP, GTP s, has
been shown to stabilize these rings and to prevent the fission reaction
(Koenig and Ikeda, 1989; Hinshaw and Schmid, 1995; Takei et al., 1995,
1996).
The hypothesis that the interaction of amphiphysin I with dynamin
I occurs in situ and is important for the function of
dynamin I is supported by a variety of studies. First, amphiphysin I
and dynamin I have a similar distribution in the brain, where both are
concentrated at synapses (Lichte et al., 1992; David et al., 1996).
Second, pools of dynamin I and amphiphysin I can be coprecipitated from
brain extracts (David et al., 1996) and undergo parallel dephosphorylation after nerve terminal stimulation (Robinson et al.,
1993; Bauerfeind et al., 1998). Third, disruption of the interaction of
the proline-rich tail of dynamin I with SH3 domains, including the SH3
domain of amphiphysin I, produces an impairment of synaptic vesicle
endocytosis at the stage of deeply invaginated clathrin-coated pits
(Shupliakov et al., 1997), i.e., the stage at which the action of
dynamin I has been implicated (Koenig and Ikeda, 1989). There is
evidence to suggest that amphiphysin I acts upstream of dynamin and is
important in its recruitment to sites of endocytosis (Shupliakov et
al., 1997).
In agreement with these results, genetic studies in yeast have
demonstrated a role of the RVS genes in endocytosis (Munn et al., 1995). In addition, yeast genetic studies also have implicated the
RVS genes in actin function and cell polarity (Munn et al., 1995; Sivadon et al., 1995). Whether a similar link exists between amphiphysin family members and actin in higher vertebrates remains unclear, although recent studies on amphiphysin II suggest pleotropic roles of this protein (Sakamuro et al., 1996; Butler et al., 1997; Leprince et al., 1997), including roles in clathrin-mediated
endocytosis and in cortical actin function (Butler et al., 1997;
Ramjaun et al., 1997).
In vitro studies on neurons of shibire
mutants of Drosophila have shown that they display an
impairment of neurite outgrowth at the restrictive temperature (Masur
et al., 1990). Furthermore, suppression of dynamin I expression by
antisense oligonucleotides in cultured hippocampal neurons and
neuroblastoma cells was found to produce a potent inhibition of neurite
formation and the collapse of growth cones (Torre et al., 1994). These
findings indicate a critical role for dynamin I in neuritogenesis,
although its mechanism of action in growth cone dynamics remains
unclear. The role of dynamin I in neurite formation may be the
consequence of an endocytotic block. However, it is possible that
dynamin I suppression also may have a negative effect on the actin
cytoskeleton. For example, transfection of a dominant negative mutant
dynamin in fibroblastic cells was found to inhibit actin dynamics at
the cell periphery (Damke et al., 1994). If amphiphysin I is a
physiological upstream partner of dynamin I, it may be expected that
amphiphysin I also may be required in growth cone dynamics. This action
would be consistent with the known role of the yeast Rvs proteins in actin function and polarized growth.
We show here that suppression of amphiphysin I by antisense
oligonucleotides has a potent inhibitory effect on neurite outgrowth, as previously shown for dynamin I suppression (Torre et al., 1994). We
also report that amphiphysin I and dynamin I are closely colocalized with each other and in turn colocalize with actin patches in growth cones of developing neurons. These findings suggest that, similar to
its yeast homologs, amphiphysin I may have pleotropic roles in
endocytosis, actin function, and cell polarity.
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MATERIALS AND METHODS |
Antibodies and reagents. The following antibodies
were raised in our laboratory: rabbit and mouse polyclonal antibodies
directed against full-length amphiphysin (David et al., 1996), rabbit
polyclonal antibodies against synapsin I (De Camilli et al., 1983a),
rabbit polyclonal antibodies against human dynamin I (DG-1) (Butler et al., 1997), and rabbit polyclonal antibodies against luminal domain of
synaptotagmin (Mundigl et al., 1995). A monoclonal antibody directed
against syntaxin 1 (HPC-1) was a kind gift from Dr. C. Barnstable (Yale
University, New Haven, CT). Monoclonal antibodies directed against
dynamin I (Hudy-1) and  -tubulin were purchased from Upstate
Biotechnology (Lake Placid, NY) and Sigma (St. Louis, MO),
respectively. MAP2 and actin antibodies were purchased from Boehringer
Mannheim (Indianapolis, IN). Rhodamine- and fluorescein-labeled phalloidin was purchased from Molecular Probes (Eugene, OR). Secondary antibodies conjugated with CY3, DTAF, and cascade blue were purchased from Jackson ImmunoResearch (West Grove, PA) and Molecular Probes.
Antisense oligonucleotides. All oligonucleotides used in
this study were unmodified phosphodiester oligonucleotides synthesized in the W. M. Keck facility at Yale University. Two amphiphysin antisense oligonucleotide constructs, termed AMAS3
(5 -GCTGCGGGTCCGGGGAGCTG-3) and AMAS4
(5-GAGCTGCGAGAGCAGAGCG-3), were designed on the basis of the
amphiphysin cDNA sequence. Control sense constructs were synthesized as
the reverse complement of the antisense oligonucleotides. An oligo
termed AMAS.3.SCR (5-GCGTGTTGCGGGCGCCAGGG-3) was
designed to contain the same content of nucleotides as AMAS3 but in a
random order. The sequences did not match any other known gene listed in the database. To facilitate the uptake of the oligonucleotides into
cells, we complexed them at a stoichiometry of 1:1 with cationic copolymer molecules (PS980) that were shown to enhance cell entry and
possibly the stability of small nucleic acid fragments (Kabanov et al.,
1995). PS980 copolymer was provided by Supratek Pharma (Montréal,
Québec, Canada). These molecules consist of a poly(ethylene oxide) and positively charged poly(spermine) segment. They
spontaneously form complexes with oligonucleotides in which the charges
of the poly(spermine) segment and DNA are neutralized. Despite the
charge neutralization, the complexes remain in solution because of the solubilizing effect of the poly(ethylene oxide). The complexes are
thought to self-assemble into uncharged micelles with cmc in
the micromolar range (Kabanov et al., 1995, 1996). Oligonucleotides were applied at the final concentration of 1.0 µM in
fresh glia-conditioned "neuronal" medium. Initially, the optimal
oligonucleotide concentration was determined by applying 0.5, 1.0, and
2.0 µM. These complexes produced suppression of the
target gene at final oligonucleotide concentrations in the range of 1 µM, which were 50-100 times lower that when
oligonucleotides were used alone (Vinogradov et al., 1994).
Hippocampal cell cultures. A monolayer of cortical glial
cells was grown as described (Banker and Cowan, 1977; Goslin and Banker, 1990). Neuronal cultures were prepared essentially as described
by Banker and Cowan (1977). Briefly, hippocampi from 18-d-old fetal
rats were dissociated by treatment with trypsin (0.25% for 15 min at
37°C), followed by trituration with a fire-polished Pasteur pipette.
Dissociated cells were plated on poly-L-lysine-treated glass coverslips in MEM with 10% horse serum at densities ranging from
3000 to 25,000 cells/cm2. After 4 hr the coverslips
were transferred, face down, to dishes containing the monolayer of
cortical glial cells in neuronal medium MEM (Life Technologies,
Gaithersburg, MD) without sera, supplemented with 1% HL1 (Ventrex,
Portland, ME), 2 mM glutamine, and 1 mg/ml BSA. For
treatment with sense or antisense oligonucleotides, coverslips with
freshly plated neurons were transferred to 12 well plates devoid of
glia and were incubated in glia-conditioned medium. The medium was
replaced every 12 hr with fresh oligonucleotide containing conditioned
medium. Glia-conditioned medium was prepared by adding neuronal medium
to sets of glia-containing dishes 12 hr before neuronal plating. The
medium of one of this dishes was used for each change.
Immunocytochemistry. Cultures were fixed by 4% formaldehyde
(freshly prepared from paraformaldehyde) in 0.1 M phosphate
buffer containing 0.12 M sucrose, detergent-permeabilized,
immunostained, and prepared for microscopic observation as previously
described (De Camilli et al., 1983a; Cameron et al., 1991). For triple
labeling, the primary antibodies were applied together with
fluorescein-labeled phalloidin and detected with CY3 and cascade
blue-conjugated secondary antibodies. Pictures were taken with T-MAX
100 (Kodak, Rochester, NY) film at a Zeiss Axiophot microscope
(Oberkochen, Germany) equipped with epifluorescence microscopy.
Western blot analysis and protein determination. Neurons on
coverslips were rinsed in warm Krebs'-Ringer's-HEPES buffer and then scraped off the coverslip directly into Laemmli sample buffer for
SDS-PAGE. Total protein was determined with the NanoOrange protein
assay (Molecular Probes) after solubilization of the neurons from
coverslips by exposure to 95°C for 10 min in NanoOrange protein reagent. SDS-PAGE was performed according to Laemmli (1970). Transfer of the proteins to nitrocellulose membranes (Millipore, Bedford, MA)
and Western blot analysis (Kyhse-Andersen, 1994) that used 125I-Protein A (Amersham, Arlington Heights, IL) or ECL
were performed as described.
Transferrin uptake. Hippocampal cultures were treated with
CY3-conjugated human transferrin (20 µg/ml final concentration) for
20 min at 37° in culture medium and then rinsed in the same buffer
and fixed as described above.
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RESULTS |
Amphiphysin I expression in cultured
hippocampal neurons
As a premise to the study of the properties of amphiphysin I
in growth cones of hippocampal neurons in vitro, we
investigated the pattern of expression of this protein during neuronal
development. Figure 1 shows the Western
blot analysis of homogenates from hippocampal cultures harvested after
various days in vitro (DIV). Amphiphysin I is expressed, and
its level increases ~10-fold between 3 and 25 DIV. This increase
parallels the time course of neurite outgrowth and synaptogenesis and
is consistent with an important role of amphiphysin I in neurite
elongation and presynaptic function in hippocampal neurons.
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Figure 1.
Amphiphysin I expression is upregulated during the
development of hippocampal neurons in vitro. Shown is a
Western blot analysis (using 125I-Protein A as an antibody
detector) of homogenates from cultures after 3, 11, and 25 d
in vitro. This time course correlates with the growth of
neurites and the formation of synapses in these cultures. Equal amounts
of total protein were added to each lane. Amphiphysin immunoreactivity
was detected by autoradiography (top) or measured in a
gamma counter (bottom).
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Immunofluorescence staining of mature cultures rich in synaptic
contacts revealed that both amphiphysin I and dynamin I are concentrated along the surface of perikarya and dendrites, where they
form "hot spots" that have the localization typical of presynaptic compartments (Fig. 2a,b).
Accordingly, these sites correspond to hot spots of immunoreactivity
for synapsin I, a synaptic vesicle-associated protein (De Camilli et
al., 1983a,b) (Fig. 2c,d and e,f).
However, whereas synapsin I is strictly confined to well separated
puncta representing synaptic vesicle clusters (Fletcher et al., 1991), amphiphysin I (Fig. 2e) and dynamin I (see Fig.
2b) are present also in connecting axonal segments, possibly
reflecting a soluble pool of these proteins (compare Fig. 2e
with f). This pool is clearly visible in cultures
double-stained for the dendritic marker MAP2 and for amphiphysin I
(Fig. 2g,h). In conclusion, the distribution of the
amphiphysin I and dynamin I in "mature" neuronal cultures is very
similar to the previously reported distribution of these proteins in
brain tissue (McPherson et al., 1994; David et al., 1996) and validates
the use of hippocampal neurons for the study of the function of
amphiphysin I on neuronal development.
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Figure 2.
Concentration of both amphiphysin I and dynamin I
at presynaptic contacts in mature hippocampal cultures (2-3 weeks
in vitro). a, b, Double
immunofluorescence for amphiphysin I and dynamin I. Both proteins are
concentrated at "hot spots" along the perikaryal and dendritic
surface of the neuron shown in the field. c-f, Double immunofluorescence for amphiphysin I and for the synaptic vesicle marker synapsin I, demonstrating that amphiphysin I immunoreactivity is
not restricted to synaptic vesicle clusters but extends to interconnecting axonal segments. g, h, Double
immunofluorescence for amphiphysin I and for the dendritic marker MAP2
further illustrating that low levels of amphiphysin are also present in
nonsynaptic regions of axons (arrow). Scale bar: for
a-d, g, h, 18.6 µm; for e, f, 9.3 µm.
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Amphiphysin I is concentrated in growth cones of
developing neurites
The distribution of amphiphysin I in isolated developing neurons
[stage 3 neurons according to the classification of Dotti et al.
(1988)] is illustrated in Figure 3,
which shows cultures stained for actin, amphiphysin I, and tubulin.
Amphiphysin I is detectable in the perikaryon, dendritic tree, and
distal axon. It is concentrated particularly at the tips of both axons
(Fig. 3b) and dendrites (Fig. 3b,e), which also
are enriched in actin (Fig. 3a,d). Similar observations were
made for dynamin I (data not shown).
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Figure 3.
Distribution of amphiphysin I in cultured
hippocampal neurons developing in isolation (stage 3 neurons).
a-c, Low-power view of a neuron triple-stained for
F-actin (TRITC phalloidin), amphiphysin (DTAF), and tubulin (cascade
blue). Amphiphysin I is concentrated in the perikaryon and distal
actin-rich portion of the axon and dendrites. Amphiphysin I (a
neuron-specific protein) is absent from two glial cells visible in the
field (arrows). d, e,
High-power view of the somatodendrite region of a neuron double-stained
for F-actin (TRITC phalloidin) and amphiphysin I (DTAF). Note the striking colocalization of amphiphysin and actin in the growth cones of
all processes. Scale bar: for a-c, 72 µm; for
d, e, 26.3 µm.
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High-power observation revealed that the localization of amphiphysin I
and dynamin I is virtually superimposable in both dendritic and axonal
growth cones. This is illustrated clearly in Figure 4, which shows the localization of the
two proteins in one of the giant axonal growth cones that occasionally
are found in hippocampal cultures (Mundigl et al., 1993). The large
flat geometry of these growth cones makes them especially suitable for
high-resolution light microscopy. Amphiphysin I and dynamin I are
concentrated particularly in patches along the rim of the growth cone
in the peripheral region that separates the organelle-rich core of the growth cone from the filopodia (Mundigl et al., 1993). These patches precisely coincide with patches of filamentous actin, as demonstrated by counterstaining with fluorescent phalloidin, a selective marker for
F-actin (Fig. 5).
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Figure 4.
Distribution of amphiphysin I in the giant growth
cone of an isolated neuron. A, B,
High-power view of a giant growth cone double-stained for amphiphysin
and for dynamin I. Amphiphysin I and dynamin I have a virtually
identical localization and are concentrated in patches along the rim of
the growth cone. Scale bar, 17 µm.
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Figure 5.
Colocalization of a pool of amphiphysin I with
F-actin in giant growth cones. Shown is double staining with
anti-amphiphysin antibodies and phalloidin. The patches of intense
amphiphysin immunoreactivity at the rim of the growth cone coincide
with patches of F-actin. Scale bar, 17 µm.
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Suppression of amphiphysin I expression inhibits
neurite development
We next used an antisense oligonucleotide approach to test the
effect of the suppression of amphiphysin I expression on neurite outgrowth. To this aim we applied a new nucleic acid delivery system
based on a polycationic amphipathic polymer that forms complexes with
the negatively charged nucleic acid (Vinogradov et al., 1994). This
method, which was never used previously on neurons, allows for the use
of a substantially lower concentration of oligonucleotides than
standard methods (50- to 100-fold lower), thus eliminating possible
side effects and increasing the specificity. As shown in Figure
6a, a treatment of freshly
plated neurons with antisense oligonucleotides for 48 hr reduced the
levels of amphiphysin I by ~90% when compared with sense
oligonucleotide-treated control cultures. No effect was observed by
using a random oligo of the same nucleotide composition as the
antisense oligo (data not shown). The treatment with antisense
oligonucleotides did not affect the overall protein expression pattern
of the neurons, as revealed by silver staining (Fig. 6b) and
by blotting for the housekeeping proteins tubulin and actin (Fig.
7A). These observations speak against a nonspecific cytopathic effect of the oligonucleotides on
these cultures. A barely detectable decrease was observed in the levels
of the synaptic vesicle protein synaptotagmin and of dynamin I (Fig.
7B), possibly reflecting a decrease in the state of
differentiation of the neurons in the antisense-treated cultures. The
effect of antisense treatment was reversible, because growth of
neurites was observed after withdrawal of the antisense
oligonucleotides.
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Figure 6.
Inhibition of amphiphysin I expression by
antisense oligonucleotides. a, Freshly plated
hippocampal neurons were treated for 48 hr with a 1.0 µM
concentration of either sense or antisense oligonucleotides, and then
equal protein aliquots of the two cultures (4 µg) were
subjected to SDS-PAGE (7.5%) and Western blot analysis, using
125I-Protein A. An autoradiogram of the blot and the
quantification of band radioactivities performed in a gamma counter are
shown. b, Silver staining of gel lanes identical to
those used for the Western blots, demonstrating that the overall
protein expression pattern is not affected by antisense treatment.
Proteins (5 µg) were separated on a 5-15% gradient SDS-PAGE and
were silver-stained.
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Figure 7.
Western blot analysis of the expression levels of
specific proteins in sense- and antisense-treated hippocampal neurons.
A, Immunolabeling for amphiphysin tubulin and actin
demonstrating that, despite the drastic effect on amphiphysin
expression, there is no change in actin or tubulin expression.
B, Immunolabeling for dynamin I and synaptotagmin
demonstrating a slight decrease in the level of these two
proteins.
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The effect of antisense treatment on neuronal morphology was assessed
by staining with antibodies directed against markers of the
cytoskeleton (tubulin and actin) and a marker of the neuronal plasmalemma (syntaxin I) (Galli et al., 1995). Sense-treated neurons were indistinguishable from untreated cells. They had a long axonal process and several dendrites (Fig.
8a,c,d). Antisense-treated neurons had a very different morphology. In most cases they did not
develop any process or had only a few short processes (Figs. 8b,e,f,
9d-f). Both axonal
(Fig. 8b) and dendritic (Fig. 9) growth cones were
completely absent. Furthermore, a profound disruption of the actin
cytoskeleton was observed. Although in control (see Fig. 3) and
sense-treated cultures (Figs. 8d, 9a,b) the actin and amphiphysin I were concentrated at the tips of the dendritic and
axonal growth cones, treatment with antisense oligonucleotides induced
the collapse of the bulk of F-actin (Figs. 8f,
9d). The organization of the tubulin cytoskeleton did not
seem to be affected (Figs. 8b, 9f) beyond
the obvious modification caused by altered cell shape.
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Figure 8.
Amphiphysin I antisense oligonucleotides inhibit
neurite development in hippocampal neurons in vitro.
Neurons were treated for 48 hr with 1.0 µM sense
(a, c, d) or antisense (b, e, f)
oligonucleotides and then processed for anti-tubulin (a,
b) immunofluorescence to reveal the overall cell morphology.
Sense and antisense neurons also were double-labeled for syntaxin
(c, e) and actin (d,
f). Scale bar, 73 µm.
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Figure 9.
Amphiphysin I antisense oligonucleotide
treatment induces a collapse of the actin cytoskeleton. Shown is triple
immunofluorescence labeling for F-actin (TRITC phalloidin), amphiphysin
I (DTAF), and -tubulin (cascade blue). Actin and amphiphysin I
accumulate at the tips of the growth cones in sense-treated cells
(a, b). Incubation with antisense
oligonucleotides results in a collapse of F-actin into tangles
primarily concentrated at one pole of the cell
(d). The residual levels of amphiphysin are
barely detectable (e), whereas the structural
organization of the tubulin network appears normal (c,
f) beyond changes caused by different cell shape.
Note the lack of actin at the tips of short cell processes visualized
by tubulin immunostaining. Scale bar, 27 µm.
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Despite these changes, rhodamine-labeled transferrin, which is known to
be internalized via clathrin-coated vesicles from the surface of
perikarya and dendrites (Cameron et al., 1991), was taken up and
concentrated in perinuclear spots in both sense- and antisense-treated
neurons (Fig. 10). Likewise,
fluid-phase uptake of horseradish peroxidase and uptake of wheat germ
agglutinin was similar in sense- and antisense-treated cultures (data
not shown).
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Figure 10.
Uptake of fluorescent transferrin in sense- and
antisense-treated hippocampal neurons. Transferrin uptake is not
inhibited in the antisense-treated neuron. Note the presence of
numerous dendrites in the sense- but not in the antisense-treated
neurons.
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DISCUSSION |
Our results demonstrate that amphiphysin I plays a key role not
only in the function of the mature presynaptic compartment but also in
the development of axons. We report that the suppression of amphiphysin
I expression in developing hippocampal neurons by antisense
oligonucleotides has a potent inhibitory effect on neurite outgrowth
and produces a collapse of growth cone. This phenotype mimics the
effect previously shown to be produced by the suppression of dynamin I
expression (Torre et al., 1994), thus providing further support to the
hypothesis that the functions of amphiphysin I and dynamin I are
interrelated (David et al., 1996). This hypothesis is strengthened by
the close colocalization of dynamin I and amphiphysin I at all
developmental stages.
The powerful inhibition of amphiphysin I expression was achieved by the
use of a novel oligonucleotide delivery system based on a cationic
compound that spontaneously forms water-soluble complexes with nucleic
acids under physiological conditions (Vinogradov et al., 1994). This
system allows for a very substantial reduction (one to two orders of
magnitude) in the concentration of oligonucleotides necessary to
inhibit protein expression. As shown here, this oligonucleotide delivery system is very effective on neurons.
The mechanism by which amphiphysin I and dynamin I suppression block
neurite outgrowth remains unclear. It is plausible that this effect may
be accounted for, at least in part, by inhibition of membrane recycling
at the cell surface. However, we did not observe a block in fluid-phase
endocytosis nor of transferrin internalization by light microscopy
cytochemistry. Because of the early state of differentiation of the
antisense-treated neurons, synaptic vesicle endocytosis could not be
assessed properly. An attractive possibility is that suppression of
neurite outgrowth in amphiphysin I- and dynamin I-depleted neurons may
reflect, at least in part, a role of amphiphysin I and dynamin I
(either directly or via signaling cascades) in the function of the
actin cytoskeleton.
An effect of dynamin I on actin function has been suggested by the
block in the dynamics of cortical actin produced in fibroblasts by
transfection of a mutant dynamin defective in GTP binding and hydrolysis (Damke et al., 1994). A relationship between amphiphysin I
function and actin is suggested by studies on the yeast homologs of
amphiphysin I, the Rvs167 and Rvs161 proteins (Crouzet et al., 1991;
Sivadon et al., 1995). Rvs167 has a domain structure similar to
amphiphysin, whereas Rvs161 represents a truncated form of amphiphysin/Rvs167 and includes only the coiled-coil NH2 terminal domain (David et al., 1994). Both of these yeast proteins have been
shown by genetic studies to be involved in endocytosis (Munn et al.,
1995). In addition, both proteins have been linked to actin function by
a variety of findings, which include genetic interaction between
RVS161 and the actin gene (ACT1), effects of
RVS genes disruption on cell polarity, cell morphology, and localization of actin patches (Munn et al., 1995; Sivadon et al., 1995). Furthermore, an interaction between Rvs167 and yeast actin has
been demonstrated by a two-hybrid screen, although it remains to be
proven that this interaction is direct (Amberg et al., 1995). So far
attempts to demonstrate a direct interaction between amphiphysin I with
either G-actin or F-actin have proved unsuccessful (our unpublished
results). However, the close colocalization of a pool of amphiphysin I
(and dynamin I) with actin patches demonstrated here supports a link
between amphiphysin family members and the actin cytoskeleton.
Amphiphysin may interact indirectly with actin via other proteins
concentrated at actin patches. The other major neuronal binding
partner, besides dynamin, for the SH3 domain of amphiphysin I is the
inositol 5-phosphatase synaptojanin (McPherson et al., 1996). The
property of synaptojanin to cleave phosphoinositides (McPherson et al.,
1996; Woscholski et al., 1997), which are potent regulators of actin
function (Janmey, 1994), may reflect another indirect relationship
between amphiphysin and actin.
The establishment of axonal polarity in neurons is controlled at least
partially by the same fundamental mechanisms that control polarity in
yeast (Luo et al., 1994, 1997; Drubin and Nelson, 1996). It is
therefore of interest that the suppression of Rvs/amphiphysin function
impairs polarity in yeast and neurite extension in neurons. Both of
these effects may be achieved by similar mechanisms involving the actin
cytoskeleton. We note that an inhibition of neurite outgrowth also was
observed after treatment of neuronal hippocampal cultures with
antisynapsin II antisense oligonucleotides (Ferreira et al., 1994).
Synapsin is an actin-binding protein that participates in the synaptic
vesicle cycle (De Camilli et al., 1990). Thus, a dual function in
neuritogenesis and presynaptic function appears to be a property shared
by a variety of cytosolic presynaptic proteins.
A possible connection between amphiphysin function and the cortical
actin cytoskeleton raises the question of whether the function of
amphiphysin I in endocytosis involves actin. Studies in yeast have
demonstrated that many other genes, besides RVS genes, play a dual role
in endocytosis and actin function. For example, the genes END3,
END4, END5, END7, and SAC6 are required both for
endocytosis and for correct actin localization (Kübler and
Riezman, 1993; Raths et al., 1993; Benedetti et al., 1994; Munn et al.,
1995). One such gene, END7, is the actin gene itself (ACT1) (Munn et al., 1995). SAC6 and
END4 are similar to the mammalian actin-binding proteins
fibrin and talin, respectively (Adams et al., 1995; Brower et al.,
1995; Munn et al., 1995). Conversely, it was found recently that
mutations in one of the actin motors, the myosin protein Myo5, produces
endocytosis defects (Geli and Riezman, 1996). Several studies have
shown that even in mammalian cells actin plays an important role in
endocytosis. An involvement of actin is well documented in fluid-phase
endocytosis (Schmalzing et al., 1995; Lamaze et al., 1996), but growing
evidence suggests that actin participates in clathrin-mediated
endocytosis also (Gottlieb et al., 1993; Evangelisti et al., 1995;
Durrbach et al., 1996). Further studies of amphiphysin I and of the
amphiphysin protein family may help to shed new light on growth cone
dynamics and on the molecular mechanisms implicated in the still
elusive connection between actin and endocytosis.
| |
FOOTNOTES |
Received June 26, 1997; revised Oct. 14, 1997; accepted Oct. 15, 1997.
This work was supported by grants from the Donaghue Foundation, the
Human Frontier Science Program, and the National Institutes of Health
(CA46128) to P.D.C.; by a postdoctoral fellowship from the Muscular
Dystrophy Association to V.I.S.; and by the United States Army Medical
Research and Development Command to C.D. We thank Laurie Daniell for
outstanding technical support. We also thank the W. M. Keck
Facility at Yale University for oligonucleotide synthesis.
Correspondence should be addressed to Dr. Pietro De Camilli, Department
of Cell Biology, Howard Hughes Medical Institute, Yale University
School of Medicine, Boyer Center for Molecular Medicine, 295 Congress
Avenue New Haven, CT 06510.
| |
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Abstract
Introduction
