1 Institute of Neuroscience, Chinese Academy of Sciences, Shanghai 200031,
China
2 Department of Biochemistry and Molecular Neuroscience Center, Hong Kong
University of Science and Technology, Clear Water Bay, Hong Kong, China
3 Division of Neurobiology, Department of Cell and Molecular Biology, University
of California, Berkeley, CA 94720, USA
* Author for correspondence (e-mail: boip{at}ust.hk)
Accepted 28 July 2005
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SUMMARY |
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Key words: Axon guidance, Neurite outgrowth, PI3-kinase, Rac1, Calcium signaling
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Introduction |
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In addition to motoneurons, agrin is expressed by all neuronal populations
in the central nervous system (CNS), and is implicated to have potential
functions in the formation and maturation of central synapses
(Cohen et al., 1997;
Kroger and Schroder, 2002
;
O'Connor et al., 1994
;
Rupp et al., 1991
). In
addition, the prominent expression of agrin in neurons during axonal growth
prior to synapse formation suggests that it may have a presynaptic function,
e.g. in regulating axon extension. Consistent with this notion, aberrant
arborization of motor axon terminals was observed in the agrin knockout mice
(Gautam et al., 1996
). Cell
culture studies have also shown that neurite extension was inhibited by
various isoforms of agrin (Bixby et al.,
2002
; Campagna et al.,
1995
; Mantych and Ferreira,
2001
).
How agrin exerts its action on developing neurons is beginning to be
explored. Treatment of primary neuronal cultures with agrin leads to the
activation of cAMP-response element binding protein
(Ji et al., 1998) and the
expression of c-fos (Hilgenberg
et al., 1999
). To initiate its action on neurons, agrin is likely
to first activate a cell-surface receptor. Evidence for agrin-binding sites on
the surface of central neurons has recently been reported
(Burgess et al., 2002
;
Hoover et al., 2003
), although
the identity of the receptor and the immediate cytoplasmic transduction events
triggered by agrin binding to the neuronal surface remain unknown. At the
NMJs, postsynaptic MuSK activation is known to mediate the effect of agrin in
inducing AChR clusters. Unlike agrin, the expression of MuSK in mammals
appears to be largely restricted to muscle, although MuSK transcripts could
also be detected in neural tissues in lower vertebrates
(Fu et al., 1999
;
Ip et al., 2000
;
Valenzuela et al., 1995
).
Thus, the possibility exists that the action of agrin on neurons may be
mediated through a receptor tyrosine kinase that is homologous to MuSK, in a
manner similar to that in the muscle cell.
In this study, we have examined the effects of agrin on neurite extension and steering using developing spinal neurons prepared from Xenopus embryos. We report that agrin inhibits neurite outgrowth in a dose-dependent manner and that a gradient of agrin results in Ca2+-dependent repulsive growth-cone turning. Furthermore, we show that the agrin-induced neuronal response depends upon the activity of Rac1. Taken together, our findings suggest that agrin regulates neurite extension and, more importantly, provide the first demonstration of a novel role of agrin in the steering of growing axonal terminals.
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Materials and methods |
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Neurite extension and growth cone turning assay for Xenopus spinal neurons
Fast-growing neurons were used for the neurite extension and growth-cone
turning assay. Extending neurites were captured at different time intervals
with a time-lapsed CCD (charge-coupled device) camera (TK-C1381; JVC,
Yokohama, Japan) attached to a phase-contrast microscope (CK-40, Olympus,
Tokyo, Japan) and analyzed using Scion Image programs. Only neurons with a
neurite extension rate of more than 5 µm/hour prior to drug treatment were
included for analysis. Neurite extension rate was normalized by comparing the
extension rates of the neurons before and after the addition of drugs for the
indicated periods. The growth-cone turning assay was carried out as described
(Song et al., 1997). Briefly,
microscopic gradients of drugs were produced with a micropipette placed 100
µm away from the center of the growth cone of an isolated neuron, at an
angle of 45° with respect to the initial direction of neurite extension
(indicated by the last 10 µm segment of the neurite). The turning angle was
defined as the angle between the original direction of neurite extension and a
straight line connecting the positions of the growth cone at the beginning and
the end of the 1-hour period. Theoretical analysis and direct measurements of
the gradient using fluorescent dyes have shown that, at a distance of 100
µm from the pipette tip, the concentration gradient across the growth cone
(typical width 10 µm) is in the range of 5-10%, and the average
concentration at the growth cone is about 103-fold lower than that
in the pipette. Microscopic images of neurites were captured and stored using
Scion Image programs as previously described
(Yuan et al., 2003
). To
determine the total length of neurite extension, the whole trajectory of the
neurite at the end of the 1-hour period was measured with a digitizer. Only
those growth cones with a net extension of more than 5 µm over the 1-hour
period were included in the analysis of turning angles.
All experiments were carried out at room temperature in modified Ringer's solution (140 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2 and 10 mM HEPES, pH 7.4). Agrin was obtained from R&D Systems and prepared in 1xPBS containing 50 µg/ml of bovine serum albumin at pH 7.4. Data were reported as means±s.e.m.; statistical significance was analyzed by Student's t-test or one-way ANOVA.
cDNAs encoding MuSK mutant and Rac1 GTPase, and microinjection into Xenopus embryos
A MuSK cDNA fragment that lacked the tyrosine kinase domain was subcloned
into pcDNA1 tagged with the Fc region of Ig to generate EC-MuSK
(Yang et al., 1997).
FITC-dextran and EC-MuSK cDNA were microinjected into one or two blastomeres
of 2- or 4-cell-stage embryos with an Eppendorf transjector 5246 (Eppendorf,
Hamburg, Germany). Injected embryos were incubated in 10% Ringer's solution at
room temperature (20-22°C) for 24 hours prior to culture preparation. The
green fluorescence of FITC-dextran was used to identify the injected progeny
cells as previously described (Ming et
al., 1999
). The cDNA construct encoding N17 dominant-negative Rac1
(DN-Rac1) fused with GFP was subcloned into pCS2 (a gift from D. Turner,
University of Michigan, Ann Arbor, MI) at the StuI site. DN-Rac1
(generously provided by G. Bokoch, Scripps Research Institute, La Jolla, CA,
USA) is a competitive inhibitor of Rac GTPase that binds irreversibly to
guanine nucleotide-exchange factors, which are upstream regulators of GTPases.
The plasmids were purified using Endofree Plasmid Maxi kit (Qiagen, Hilden,
Germany). The final concentration of cDNAs for microinjection was 0.2
µg/µl for DN-Rac1 and 0.5 µg/µl for GFP and EC-MuSK, and total
amounts of 1.5 ng and 5 ng were injected, respectively.
Western blot analysis and GTPase activation assay
Expression of injected constructs was confirmed using western blot
analysis. Five Xenopus embryos (stage 22-24) were collected and
homogenized in 0.2 ml of lysis buffer (0.1% SDS, 1% Nonidet P-40, 1% glycerin,
50 mM HEPES, pH 7.4, 2 mM EDTA and 100 mM NaCl) by sonication. The homogenates
were centrifuged at 13,000 g for 5 minutes. The supernatant
was mixed with equal volume of 1,1,2-trichlorotrifluoroethane and centrifuged
again to remove the yolk. The blots were incubated with antibodies against
EGFP (polyclonal, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and
Rac1 (monoclonal, 1:1000; Upstate, Charlottesville, VA, USA) at 4°C
overnight. Chemiluminant detection was performed using the Supersignal kit
(Pierce, Rockford, IL, USA).
GTPase activity was measured using a Rac/Rho activation assay kit (Upstate). Briefly, cultured cerebellar granule neurons from postnatal day 6 to 8 (P6-P8) were treated with agrin, the cells were washed with ice-cooled PBS, lysed at 4°C and incubated either with Pak1-PBD (Pak-binding domain of Rac1 and Cdc42) agarose, or Rhotekin-binding Sepharose beads with constant rocking at 4°C. The proteins bound to the beads were washed three times with lysis buffer at 4°C, eluted in SDS sample buffer, and analyzed for bound Rac1 or Rho by western blotting using antibodies against Rac1 or Rho. GTPase activity was quantified by densitometry analysis of the blots.
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Results |
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To further confirm the specificity of the inhibitory response to agrin, an
alternative approach of expressing a fusion protein (EC-MuSK) comprising the
extracellular and transmembrane domains of MuSK fused to the Fc region of an
immunoglobulin was utilized. A similar strategy of using truncated forms of
receptor tyrosine kinases was shown to be effective in neutralizing the
activity of their cognate ligands (Croll et
al., 1998; McMahon et al.,
1995
). We expressed the fusion protein in Xenopus spinal
neurons by injecting FITC-dextran together with a cDNA construct encoding
EC-MuSK into the blastomeres of stage 22 Xenopus embryos. In cultures
of dissociated spinal neurons, the green fluorescence provided a reliable
marker for identifying progeny cells derived from injected blastomeres (data
not shown) (Alder et al.,
1995
). Interestingly, overexpression of EC-MuSK in these neurons
not only blocked agrin-induced repulsive growth-cone turning, but apparently
converted repulsion into attraction (Fig.
1D,E).
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Dependence of agrin-induced growth-cone turning on Ca2+ and PI3-kinase
Both Ca2+and phosphoinositide 3-kinases (PI3-kinase) play key
roles in the signaling of axon guidance
(Ming et al., 1999; Nishiyama,
2003). In Xenopus spinal neurons, growth-cone turning triggered by
netrin 1, brain-derived neurotrophic factor (BDNF) and myelin-associated
glycoprotein requires both Ca2+ signaling and PI3-kinase activity
(Ming et al., 1997
;
Wong et al., 2002
;
Yuan et al., 2003
). Consistent
with a previous report (Zheng et al.,
1996
), we observed that neurons cultured in Ca2+-free
solution (CFS) exhibited a higher neurite extension rate
(Fig. 3A,C). Unexpectedly,
growth cones of neurons grown in CFS showed a marked attractive turning
response towards the source of agrin (Fig.
3A). Moreover, Ca2+ release from internal stores also
appeared to be necessary, as depletion of Ca2+ stores by
pre-incubating the neurons with thapsigargin (TG) blocked the agrin-induced
turning response (Fig. 3B,C).
Taken together, these results indicate that agrin-induced repulsive growth
cone turning depends on both extracellular Ca2+ and internal
Ca2+ store. Treatments with PI3-kinase specific inhibitors,
wortmannin or LY294002, also abolished agrin-induced repulsion
(Fig. 4A,B). Taken together,
these results indicate that, like the property of some axon guidance factors
[the `group I' (Ming et al.,
2002
; Ming et al.,
1999
)], agrin-induced repulsive growth-cone turning is dependent
on both Ca2+ and PI3-kinase.
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Discussion |
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The calcium ion is a key intracellular messenger in regulating growth-cone
extension (Gomez and Spitzer,
1999; Takei et al.,
1998
) and steering (Hong et
al., 2000
; Zheng,
2000
). Similar to that observed for c-fos induction in
neurons and AChR aggregation on cultured myotubes upon agrin treatment, there
is a requirement for Ca2+ in neurite extension and growth-cone
turning initiated by agrin. We also found that Ca2+ derived from
both the extracellular space and intracellular stores is required for
mediating the effect of agrin. Moreover, we observed that neurons incubated in
CFS displayed a higher rate of neurite extension. This may be due to the
removal of Ca2+ transients in growth cones, which has been reported
to inhibit neurite extension (Lautermilch
and Spitzer, 2000
). In addition, a gradient of agrin induced
growth-cone attraction instead of repulsion in CFS. Depleting intracellular
Ca2+ stores by the pre-incubation of neurons with thapsigargin
blocked the agrin-induced turning response. This finding underscores the
importance of intracellular Ca2+ in axon guidance signaling. For a
variety of surface receptors for mammalian growth factors, PI3-kinase is a
crucial component of the initial cytoplasmic signaling pathways. The synthesis
of the lipid product of PI3-kinase has been implicated in the rearrangement of
the actin cytoskeleton, through the activation of the small GTP-binding
protein Rac1 (Hawkins et al.,
1995
; Kundra et al.,
1994
; Nobes et al.,
1995
; Wennstrom et al.,
1994
). In the present study, we showed that pre-incubation of
Xenopus spinal neurons with PI3-kinase inhibitors abolished
agrin-induced growth-cone turning. Moreover, agrin inhibited IGF1-induced
phosphorylation of Akt, a downstream target of PI3-kinase (data not shown).
Taken together, our findings suggest that the interaction of agrin with its
receptor expressed at the neuronal surface may regulate the cytoplasmic
PI3-kinase, which in turn modulates the growth-cone extension.
|
Although there is ample evidence of an agrin-dependent signaling pathway in
neurons (Hilgenberg et al.,
1999; Ji et al.,
1998
; Karasewski and Ferreira,
2003
), little is known about the identity of the neuronal agrin
receptor. Recently, the domain of agrin that binds to its receptor in neurons
was identified (Burgess et al.,
2002
; Hoover et al.,
2003
). Early studies on the expression profile of a component of
agrin receptor complex, MuSK, in mammalian species indicate that it is largely
restricted to skeletal muscle (Valenzuela
et al., 1995
). However, we have subsequently reported that MuSK
transcripts could be detected in the developing neural tube and eye vesicles
of Xenopus, and the postnatal cerebellum of chicken
(Fu et al., 1999
;
Ip et al., 2000
). To date, the
agrin receptor in the CNS was unidentified. It is noteworthy that, in the
present study, overexpression of EC-MuSK converts the repulsive agrin-induced
growth-cone turning to an attractive property. Interestingly, a MuSK homolog
was recently reported in zebrafish and suggested to be involved in axonal
pathfinding (Zhang et al.,
2004
).
Our findings on the ability of agrin to induce growth-cone turning imply
that this molecule may function as an axon guidance molecule in development.
Similar to axon guidance cues such as BDNF and netrin 1, the turning response
induced by agrin also requires both Ca2+ and PI-3 kinase. The
action of many guidance cues on growth cones can be `switched' between
attraction and repulsion in a manner that depends on the level of cytosolic
cyclic nucleotides (Jones and Werle,
2004), or the developmental stage
(Hoch et al., 1993
). It would
be of interest to determine whether the switch from repulsion to attraction
found for agrin in the present study is due to similar cytoplasmic mechanisms.
As agrin is secreted by motoneuron nerve terminals during the synaptogenesis
of NMJs, our findings suggest that these secreted agrin molecules might play a
role in shaping the pattern of motor axonal terminal arbors through their
action on the growth cones.
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ACKNOWLEDGMENTS |
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