Article |
Address correspondence to Martin A. Smith, Dept. Anatomy and Neurobiology, JISH, Rm. 110, University of California, Irvine, Irvine, CA 92697. Tel.: (949) 824-7079. Fax: (949) 824-1105. E-mail: masmith{at}uci.edu
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Abstract |
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Key Words: -dystroglycan; agrin; agrin receptor; synapse; neuromuscular junction; neuron
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Introduction |
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Native agrin is an 400-kD heparan sulfate proteoglycan assembled on an
200-kD polypeptide backbone characterized by multiple cysteine-rich domains homologous to other ECMs and secreted proteins (Fig. 1). Structural determinants required for agrin's AChR clustering activity are localized to its 95-kD COOH-terminal half distinguished by the presence of four EGF repeats and three laminin-like G domains (Ferns et al., 1993). Relatively little is known about the properties of the EGF repeats, but functions of the G domains are better understood. For example,
-dystroglycan, a component of the dystrophin-associated glycoprotein complex expressed on the surface membranes of skeletal muscle fibers, binds to a region within the G1/G2 domain (Gesemann et al., 1996; Hopf and Hoch, 1996). However, neither the G1 nor G2 domains are required for agrin's AChR clustering activity, which instead is a property of a 21-kD COOH-terminal region consisting of the G3 domain and the alternatively spliced z site (Gesemann et al., 1995, 1996). The receptor that mediates agrin-induced clustering of AChRs in muscle is a complex consisting of a muscle-specific kinase (MuSK) and an as yet to be identified myotube-associated specificity component (MASC; Glass et al., 1996). Thus, structural elements sufficient to bind and activate the MuSKMASC receptor are present within 21 kD of agrin's COOH terminus. However, the observation that the AChR clustering activity of the 21-kD fragment is markedly lower than the 95-kD fragment (Gesemann et al., 1995) suggests the G1/G2 domains and/or EGF repeats also contribute to agrin's activity, either by stabilizing the structure of the active site or binding to a component on the muscle cell surface.
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Little is known about the receptor(s) that might mediate agrin's effects in neurons. However, using the immediate early gene c-fos as a reporter, we have identified an agrin-dependent signaling pathway in central nervous system (CNS) neurons, providing evidence that a neuronal receptor for agrin does exist (Hilgenberg et al., 1999). Agrin signaling in neurons shares several biochemical similarities with that in muscle, including concentration dependence, requirement for extracellular Ca2+, and inhibition by heparin. However, unlike AChR clustering, which is exquisitely sensitive to splicing at the z site (Ferns et al., 1992; Ruegg et al., 1992; Gesemann et al., 1995), agrin z+ and z- isoforms are equally potent activators of the agrin signal pathway in neurons (Hilgenberg et al., 1999). To learn more about the nature of the neuronal receptor for agrin, we analyzed the functional properties of deletion mutants derived from the 95-kD COOH-terminal fragment used in our original works. Our results provide evidence for a novel neuronal receptor for agrin concentrated at synapses formed between CNS neurons.
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Results |
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Rat rC-Agz0 and rC-Agz8 induce a neuron-specific increase in Fos expression (Hilgenberg et al., 1999). To confirm the properties of the corresponding mouse constructs, 12-d-old cortical cultures were treated with 1 nM purified mouse C-Ag95z0 or C-Ag95z8, and were then double labeled with antibodies against Fos and either microtubule-associated protein 2 (MAP2) or glial fibrillary acidic protein (GFAP) to identify neurons and glial cells, respectively. Consistent with our previous results, treatment with either C-Ag95z0 or C-Ag95z8 caused a marked increase in Fos expression in neurons, but not nonneuronal cells (Fig. 2). Although differences in the level of Fos expression between neurons were apparent, virtually all neurons (>90%) responded to the C-Ag95z0/8 treatment. In contrast, treatment with a similar concentration of prostate serum antigen control protein expressed in the same vector had no effect on Fos levels in either neurons or glia (Fig. 2). In light of these results, we conclude that neither the myc epitope nor polyhistidine tags induce c-fos, nor do they affect the ability of the C-Ag95z0/8 sequences to do so.
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The 20-kD COOH-terminal portion of agrin is necessary and sufficient for signaling in neurons
As a first step in localizing the structural domains responsible for signaling in neurons, we took advantage of knowledge gained from previous structural analyses of agrin function in muscle (Hoch et al., 1994; Gesemann et al., 1995, 1996) and divided C-Ag95z0/8 into two fragments. The first, an 75-kD NH2-terminal fragment (Fig. 1; C-Ag20), which includes the 4 amino acid exon at the y site, has been shown to mediate agrin binding to
-dystroglycan (Gesemann et al., 1996; Hopf and Hoch, 1996). The remaining 20-kD COOH-terminal fragment contains the alternatively spliced z site (Fig. 1; C-Ag20z0 or C-Ag20z8), and is homologous to the minimal agrin fragment able to induce clustering of AChR in cultured muscle cells (Gesemann et al., 1995).
Treatment with C-Ag20, at a concentration equivalent to either a near saturating (50 pM) or supersaturating (5 nM) amount of C-Ag95z0/8, had no effect on Fos expression in cortical cultures, suggesting that the active domain is not present within the C-Ag
20 region (Fig. 3 A). Agrin binding to
-dystroglycan has been shown to modulate agrin-induced AChR clustering in muscle, and we considered the possibility of a similar function for
-dystroglycan signaling in neurons. However, coincubation with either an equal or 100-fold molar excess of C-Ag
20 had no effect on Fos expression induced by either the C-Ag95z0 or C-Ag95z8 isoforms (Fig. 3 A). Together, these data suggest that domains present within C-Ag
20 are neither required for, nor modulate, agrin induction of c-fos in neurons.
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Sequences flanking the z site are critical for agrin signaling
Agrin's AChR clustering activity is regulated by alternative splicing at the z site. However, the observation that a peptide of the 8 amino acid alternatively spliced insert is itself inactive (Gesemann et al., 1995) suggests that domains important for agrin's bioactivity in muscle include not only the z site, but also amino acids that flank it. Because inclusion of alternatively spliced exons at the z site has no effect on agrin activity in neurons, we sought to determine the role of sequences surrounding the z site. To address this question, we deleted 37 amino acids from the NH2 terminus of C-Ag20z0 to the border of the G3 domain, giving rise to a 15-kD COOH-terminal fragment, C-Ag15 (Fig. 1).
Treatment with C-Ag15 alone had no effect on the levels of Fos expression, even when present at a concentration fivefold above saturation for C-Ag20z0/8 (unpublished data). However, when added together with C-Ag95z8, C-Ag15 appeared to inhibit the c-fosinducing activity of the larger agrin fragment. To examine this effect in more detail, C-Ag15 doseresponse studies were performed in the presence of a near saturating (50 pM) concentration of C-Ag95z8. Increasing concentrations of C-Ag15 inhibited the c-fosinducing activity of C-Ag95z8 (Fig. 4 A). The curve was well fit by a nonlinear regression model for single-site competition (R2 = 0.95) predicting an IC50 of 64.45 ± 10.94 pM and close to a 1:1 agonist:antagonist molar ratio. Similar results were also obtained for competition against 50 pM C-Ag95z0 and the C-Ag20z0/8 isoforms (unpublished data).
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C-Ag20z0/8 rescues an agrin-deficient neuronal phenotype
Although induction of c-fos is a convenient reporter facilitating biochemical characterization of agrin signaling, its significance in terms of agrin function in CNS neurons is unknown. Recently, we have provided evidence that agrin plays an important role in neural differentiation by showing that agrin-deficient neurons exhibit reduced responses to glutamate both in cell culture and in vivo (Hilgenberg et al., 2002). Therefore, to learn more about the role of the 20-kD COOH-terminal region in agrin function, we tested the ability of the C-Ag20 isoforms to rescue the agrin deficiency.
When grown in normal media, levels of glutamate-induced Fos expression in agrin-deficient neurons were reduced by 70% compared with wild type. However, consistent with our earlier findings, supplementing the growth media for 2 d with a saturating concentration of either C-Ag95z8 or C-Ag95z0 significantly increased the response of agrin-deficient neurons close to wild-type levels (Fig. 5 A). Similar results were obtained when agrin-deficient neurons were grown in the presence of the C-Ag20z0/8 fragments. Compared with agrin-deficient neurons in vehicle-supplemented control media, treatment with either 5 nM C-Ag20z8 or C-Ag20z0 resulted in a significant 2.2-fold increase in glutamate response. Thus, not only do the C-Ag20z0/8 isoforms exhibit full activity in terms of their ability to induce c-fos, they are also competent to reverse the physiological deficit resulting from loss of expression of full-length agrin in the agrin-deficient cultures.
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Agrin receptors are concentrated at neuronneuron synapses
The works described so far provide evidence for a neuron-specific agrin receptor, and identify a minimal fragment of agrin capable of activating it. Next, we sought to establish the cellular distribution of the neuronal receptor for agrin using the short agrin fragments as affinity ligands. Live cortical cultures were incubated in C-Ag20z8, C-Ag20z0, or C-Ag15, and were then labeled with an antibody against the polyhistidine epitope tag to visualize bound agrin. A similar pattern of labeling was evident for all three agrin fragments and appeared as bright puncta scattered over neuronal somata and neurites (Fig. 6). Labeling was specific in that nonneuronal cells were not labeled (unpublished data), and little or no labeling was evident when either the agrin fragments were omitted or a ß-galactosidasemyc6His vector control protein was used in their place (Fig. 6). More significantly, binding of all three agrin fragments was completely blocked in cultures labeled in the presence of a large excess of rC-Ag with both "neural" rC-Agy4z8 and "inactive" rC-Agy0z0 appearing equally effective at blocking binding of either C-Ag20z isoform or C-Ag15. Precise estimates of the concentration dependence of the competition between rC-Ag and the short mouse agrin fragments are difficult to obtain using immunofluorescence. However, NIH Image analysis of the mean pixel intensities obtained from fixed exposure photomicrographs of random fields revealed labeling to be reduced by 30% in cultures coincubated with a 1:1 molar ratio of C-Ag20z8 to rC-Agy4z8, and barely detectable at 1:100 (unpublished data). Together, these results provided strong evidence that the bioactivity of the different agrin isoforms reported here is mediated through binding to a single population of agrin receptors expressed on cortical neuron cell membranes.
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Discussion |
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Initial attempts to characterize molecules that might mediate agrin-induced clustering of AChR identified -dystroglycan as a major agrin-binding protein in muscle cell membranes (Campanelli et al., 1994; Gee et al., 1994). Although it now seems unlikely to represent the "functional" agrin receptor, evidence suggests that
-dystroglycan still plays an important role in consolidation and maintenance of AChR clusters once formed (Jacobson et al., 1998, 2001; Heathcote et al., 2000). Dystroglycan is also found in mammalian brain, where its expression and synaptic localization have suggested a role in synaptic function (Tian et al., 1996; Zaccaria et al., 2001; Lévi et al., 2002). Interestingly, several biochemical properties of agrin induction of c-fos in CNS neurons, including Ca2+ dependence, inhibition by heparin, and ability to bind z+ and z- isoforms (Hilgenberg et al., 1999), are consistent with agrin binding to
-dystroglycan (Sugiyama et al., 1994; Gesemann et al., 1996). High affinity binding of agrin to
-dystroglycan is mediated by a region that includes the first two laminin G domains (Gesemann et al., 1996; Hopf and Hoch, 1996). However, C-Ag
20, which contains the G1 and G2 domains, neither induced c-fos nor inhibited the activity of the larger fragments. Conversely, both C-Ag20z0/8 isoforms, lacking domains for high affinity binding to
-dystroglycan, induced c-fos and rescued the agrin-deficient phenotype with a similar efficacy as their 95-kD C-Agz0/8 counterparts. Although these results do not rule out a role for
-dystroglycan mediating other aspects of agrin signaling,
-dystroglycan is unlikely to be a component of the agrin receptor mediating the responses measured here.
Also, we considered the possibility that the MuSKMASC complex might mediate agrin signaling in neurons. Both neural and muscle signaling pathways are activated by picomolar concentrations of agrin, are inhibited by heparin, and are Ca2+ dependent (Hilgenberg et al., 1999). Even more striking is the fact that the same minimal agrin fragment is sufficient to activate the MuSKMASC complex (Gesemann et al., 1995) and neuronal receptor; evidence that significant structural homology exists between the two receptors. However, despite these similarities, we believe the neuronal and muscle receptors are distinct. MuSK expression has not been detected in either embryonic or adult mammalian brain (Valenzuela et al., 1995). Moreover, although a critical determinant of agrin's AChR clustering activity in muscle (Ferns et al., 1992; Ruegg et al., 1992; Gesemann et al., 1995), alternative splicing at the z site has no effect on agrin's bioactivity assayed here, even in the case of the smallest active fragment where variations in structure might be expected to exert the greatest effect. The finding that the C-Ag20z0/8 fragments are fully active also contrasts with observations in muscle where the AChR clustering activity of the corresponding fragment is >100-fold lower than the 95-kD C-Ag polypeptide from which it was derived (Gesemann et al., 1995). Together with the inability of C-Ag15 to antagonize agrin induction of either c-fos or AChR clustering in muscle, these data point to a fundamental difference in the receptors that catalyze the agrin response in nerve and muscle cells.
The inhibition of c-fos induction by C-Ag15, its ability to block rescue of the agrin-deficient phenotype, distribution of binding sites, and ability of different agrin isoforms to block binding to them argue that C-Ag15 and the active agrin polypeptide fragments compete for a common binding site on the neuronal receptor for agrin. How then might C-Ag15 bind to the receptor but not activate it? One possibility is that deletion of the 5-kD NH2-terminal region may have disordered secondary or tertiary structures in the remaining G3 and COOH-terminal domain necessary for signaling. However, that such a structural change could occur without affecting the apparent affinity of C-Ag15 binding to the receptor seems improbable. Alternatively, activation of the receptor may require agrin binding to two sites, with the 5-kD NH2-terminal region of C-Ag20z0/8 targeted to the second site. The agrin receptor in muscle is made up of two components (MuSK and MASC) although agrin appears only to bind directly to MASC (Glass et al., 1996). Whereas a similar two-component model of the neuronal receptor for agrin would be consistent with our data, the fact that C-Ag15 has no effect on agrin's bioactivity in muscle suggests that, although functionally homologous to MASC, the agrin-binding component of the neuronal receptor would be structurally distinct.
Blocking agrin expression with antisense oligonucleotides inhibits synapse formation and alters synaptic function in CNS neurons (Ferreira, 1999; Böse et al., 2000). The ability of rC-Ag95z8 (but not rC-Ag95z0) to rescue changes induced by the antisense oligonucleotides suggests these effects are mediated by a loss of isoform-specific signals associated with agrin's COOH-terminal domains (Ji et al., 1998; Böse et al., 2000). These observations contrast to our own findings that, in terms of their ability to induce c-fos and rescue an agrin mutant phenotype, the bioactivity of even the shortest alternatively spliced active fragments are the same. One possible explanation for this apparent inconsistency is that different domains within the 95-kD COOH-terminal region of agrin might mediate distinct responses through specific receptors. In this regard, it is interesting to note that a recent paper showed that in addition to -dystroglycan/heparin binding, the 95-kD COOH-terminal region of agrin contains three other sites that interact with neuronal cell surface receptors (Burgess et al., 2002). Two of these sites are targeted to integrins, one of which modulates agrin's AChR clustering activity, whereas the receptor for the third site, located within 20 kD of agrin's COOH terminus, has yet to be identified. It will be interesting, in light of these observations, to determine whether C-Ag15 inhibition is limited to the bioassays used here, or whether it extends to other agrin-dependent neuronal responses.
Agrin induces expression of c-fos in CNS neurons, and mutation of the agrin gene decreases neuronal responses to excitatory neurotransmitters (Hilgenberg et al., 2002). Although neurons express multiple molecules capable of binding agrin, several lines of evidence support the conclusion that the agrin-binding sites visualized using the affinity probes are the receptors responsible for the physiologic responses to agrin reported here and earlier (Hilgenberg et al., 1999). First, the apparent affinities and potency of the various agrin fragments, in terms of their c-fosinducing activity or ability to rescue the agrin-deficient phenotype, are the same. Moreover, the ability of the smallest agrin fragment, C-Ag15, to inhibit both c-fos induction and rescue of the agrin-deficient phenotype is strong evidence for a common receptor mediating both activities. A similar profile was also apparent for the binding properties of the agrin fragments when used to visualize the distribution of the agrin receptors. Consistent with the biochemical studies, neurons were clearly labeled using agrin concentrations in the picomolar range irrespective of z site composition. Most importantly, the distribution of high density binding sites for C-Ag15 and their sensitivity to blockade by rC-Agz0/8 was indistinguishable from that observed with either C-Ag20z0/8 constructs.
Previous papers have shown that agrin is concentrated at cholinergic synapses formed between cultured sympathetic neurons (Gingras and Ferns, 2001) and GABAergic synapses in the retina (Mann and Kröger, 1996; Koulen et al., 1999). Cultured cortical neurons receive both glutamatergic and GABAergic inputs (Li et al., 1997), and because no evidence of synaptic contacts lacking agrin was found, we can extend these findings to include glutamatergic synapses as well. A similarly high correlation between agrin receptors and nerve terminals was also evident, suggesting that agrin and its receptor colocalize at synaptic sites. Together, these observations suggest that regardless of neurotransmitter phenotype, synapses are likely to be significant sites of agrin action. Consistent with these conclusions, agrin has been implicated in regulating a number of pre- and postsynaptic properties of cholinergic, glutamatergic, and GABAergic neurons (Ferreira, 1999; Böse et al., 2000; Gingras et al., 2002). Higher resolution techniques than those used here will be required to determine the agrin receptor's subcellular distribution. However, if all agrin receptors are presynaptic, then a long-range retrograde signal would be needed to account for agrin's ability to rapidly induce c-fos. Therefore, it seems likely that at least some agrin receptors, for example, those at or near the neuronal soma, are postsynaptic.
At the neuromuscular junction, agrin is anchored to the synaptic basal lamina by a laminin-binding domain present within its NH2 terminus (Denzer et al., 1995). However, the mechanism controlling agrin's spatial organization in neurons, and in particular, its concentration at synaptic sites, is unknown. Neuronneuron synapses lack a basal lamina; moreover, the short NH2-terminal form of agrin (SN-agrin) expressed in CNS neurons not only lacks the laminin-binding domain, but consistent with a type II membrane protein, is oriented such that its NH2 terminus is cytoplasmic (Burgess et al., 2000; Neumann et al., 2001). That signals embedded within this cytoplasmic domain might play a role in agrin's subcellular localization also seems unlikely. Fusion of the SN-agrin NH2 terminus to YFP directs YFP to neuronal membranes, but the pattern of expression is uniform rather than concentrated at synaptic sites (Burgess et al., 2002). In light of these findings, agrin's spatial distribution would seem to be defined by interactions between its extracellular domains and candidate neuronal cell surface agrin-binding proteins such as neural cell adhesion molecules, integrins, and -dystroglycan. Clearly, the high affinity and synapse-specific pattern of expression of the neuronal receptor for agrin are also consistent with the possibility that, in addition to signal transduction, it could play a role sculpting agrin's spatial distribution. Future studies comparing the time course of agrin and agrin receptor expression and any effect C-Ag15 might have on agrin's distribution will be helpful in testing this hypothesis.
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Materials and methods |
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Agrin-deficient neuron cultures were prepared from cortices of embryonic d 1819 fetuses resulting from matings between mice heterozygous for a mutation in the agrin gene (Gautam et al., 1996). Cultures were prepared and genotyped as described previously (Li et al., 1999), and were maintained as above.
Chick muscle cultures were prepared from pectoral muscles of 1011-d-old White Leghorn chick embryos as described previously (Hilgenberg et al., 1999). Experiments were performed on 46-d-old cultures.
Expression constructs
Parent constructs, C-Ag95z0 and C-Ag95z8 (Fig. 1), encoding the soluble 95-kD COOH terminus of mouse agrin, were generated from cDNA prepared by RT-PCR of adult mouse cortex RNA using the F95/R95 primer pair (Table I) subcloned into the pGEM-T (Promega) shuttle vector and transformed into JM109-competent bacteria. Individual ampicillin-resistant colonies were picked, and C-Ag95z0- and C-Ag95z8-containing clones were identified by PCR analysis using primers F24/B2 flanking the z site. After double digestion of the BamHI and EcoRV l linker sites contained within the F95 and R95 primers, agrin inserts were gel purified and ligated into the pSecTag2B expression vector (Invitrogen) in frame with a COOH-terminal myc epitope and 6x polyhistidine tag.
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A similar strategy was used to generate C-Ag20z0/8 and C-Ag15 constructs by PCR amplification of the appropriate pGEM-T C-Agz0/8 template using either F20 (for C-Ag20z0/8) or F15 (for C-Ag15) in combination with R20. However, for more efficient expression, aliquots of the PCR reaction were ligated into the inducible bacterial expression vector pTrcHis2 (Invitrogen).
Expression and quantitation of recombinant agrin
pSecTag2B agrin vector DNA encoding either C-Agz0/8 or C-Ag20 was transfected into HEK 293T cells using LipofectAMINETM (Invitrogen) according to the manufacturer's directions. Controls were either sham transfected with LipofectAMINETM alone or with control vector encoding prostate-specific antigen (pSecTag2-PSA). Agrin constructs in the pTrcHis2 expression vector were maintained in the JM109 bacteria. The plasmid pTrcHis2-lacZ encoding ß-galactosidase was expressed as a control.
Polyhistidine-tagged agrin fragments were purified from conditioned media and bacterial extracts using the TalonTM (CLONTECH Laboratories, Inc.) metal affinity resin eluted with 200500 mM imidazole (Sigma-Aldrich) according to the manufacturer's instructions. The identity of the isolated fragments was confirmed by immunoblot analysis using RAg-1, a rabbit antiserum raised against a synthetic peptide corresponding to amino acids 18621895 conserved in all isoforms of mouse agrin. For some experiments, the elution buffer was removed by dialysis against PBS or 20 mM Tris and 250 mM NaCl, pH 8.0.
The molar concentration of each fragment was determined by comparison to a C-Ag95z0 standard prepared as follows: HEK 293T cells were transfected with pSecTag2B-C-Agz0, and were then transferred to 80% methionine-free DME containing 100 µCi/ml [35S]methionine. C-Ag95z0 present in the medium was purified over a TalonTM metal affinity resin column, and the apparent molar concentration was determined by counting aliquots of the column eluate in a scintillation counter (model LS7500; Beckman Coulter). A small correction factor was applied to account for the fraction of counts incorporated into C-Ag95z0 (≥90%) versus total counts determined by phosphorimager analysis (Molecular Dynamics, Inc.) of an aliquot of the eluate separated on an 8% SDS-PAGE gel.
The concentration of the other agrin fragments was determined by comparison to a 35S-labeled C-Ag95z0 standard in immunoblots probed with a mouse anti-myc antibody (Invitrogen) and 125I-labeled antimouse second antibody (Amersham Biosciences). The amount of 125I bound to both the standard and unknown was determined by phosphorimager analysis and, after correcting for the contribution of the [35S]methionine in the standard, the concentration of the unknown was determined from the standard curve.
Soluble 95-kD COOH-terminal fragments of rat agrin (rC-Agy4z8/y0z0) were harvested in media conditioned by transiently transfected COS-7 cells (Hilgenberg et al., 1999) and dialyzed against PBS. The concentration of the rC-Ag was estimated by comparison to a mouse agrin standard in the c-fos induction assay and by immunoblot analysis with RAg-1.
Quantitative analysis of Fos expression
Fos expression in cortical cultures was measured by in situ enzyme-linked immunoassay (Hilgenberg et al., 1999). In brief, 1114-d-old neuronal cultures were treated for 10 min with agrin or other agent diluted in NBM or PBS, then washed in cNBM and returned to the incubator for 2 h. Cultures were rinsed in PBS, fixed in ice cold 4% PFA, and blocked in PBS containing 0.1% Triton X-100 and 4% BSA (PBSTB) before being incubated in a primary rabbit antibody against Fos (Ab-2; Oncogene Research Products) and secondary goat antibody against mouse conjugated to alkaline phosphatase (Southern Biotechnology Associates, Inc.). The level of Fos expression was determined by monitoring conversion of p-nitrophenyl phosphate to a soluble yellow reaction product at 405 nm.
AChR clustering assay
46-d-old myotubes were treated with agrin overnight followed by incubation with 20 nM rhodamine-conjugated -bungarotoxin (Molecular Probes, Inc.) in culture medium for 1 h at 37°C. Cells were then fixed in 4% PFA in PBS, washed in PBS, and viewed at 200x under epifluorescent illumination on a microscope (Optiphot-2; Nikon). For each well, the mean number of AChR clusters/field was determined from counts obtained from five random fields. All counts were performed blind with respect to treatment. To facilitate comparison between experiments, the number of AChR clusters/field was normalized to the cluster density of control cultures treated with vehicle alone.
Fos immunohistochemistry
1014-d-old cortical neurons, grown on glass coverslips, were treated with agrin, fixed, and blocked as for the Fos in situ enzyme-linked immunoassay. Cells were double labeled overnight at 4°C with Ab-2 (1:200) together with either a mouse monoclonal antibody against MAP2 (SMI-52; 1:400; Sternberger Monoclonals), or GFAP (G-A-5; 1:1,000; Sigma-Aldrich) in PBSTB to identify neurons or glial cells, respectively. Bound antibodies were visualized by incubation for 2 h at RT in a mixture of fluorescein-conjugated goat antirabbit and Texas redlabeled goat antimouse secondary antibodies (Vector Laboratories) diluted 1:200 in PBSTB. Coverslips were washed in PBS, mounted in FluoromountTM (Southern Biotechnology Associates, Inc.), and examined using epifluorescent illumination.
Agrin immunohistochemistry
Live cortical cultures on glass coverslips were incubated for 30 min at 37°C in RAg-1 diluted 1:500 in NBM. Cultures were then washed in NBM, fixed and blocked as described for the Fos in situ enzyme-linked immunoassay, and were then washed and incubated overnight at 4°C with the anti-synaptophysin antibody, SVP38 (Sigma-Aldrich), diluted 1:400 in PBS containing 4% BSA (PBSB). Primary antibodies were visualized by double labeling with fluorescein-conjugated goat antirabbit and Texas redlabeled antimouse antibodies as above.
Agrin receptor immunohistochemistry
The distribution of agrin receptors was studied using agrin deletion fragments as affinity probes. Neurons, plated on glass coverslips, were washed briefly (12 min) in cold PBS containing 10 mM EDTA followed by a second wash in cold PBS alone before incubation for 15 min at 4°C with 1 pM recombinant mouse agrin in NBM. In some experiments, labeling with mouse agrin was performed in the presence of various concentrations of rat rC-Ag95y4z8 or rC-Agy0z0. Control cultures were treated with vector control protein or an equivalent volume of vehicle in which the recombinant agrin was dissolved. Cells were then washed in cold NBM, fixed for 40 min on ice in 4% PFA in PBS, then washed in PBS and blocked for 1 h in PBSB. Recombinant agrin was detected through the COOH-terminal 6x polyhistidine tag using an anti-his antibody (1:500; Invitrogen). In experiments where cells were double labeled for synaptophysin, the anti-synaptophysin antiserum (1:400) was also added at this step. Incubations were performed overnight at 4°C in PBSB, followed by labeling with fluorescein-conjugated goat antimouse and Texas redlabeled antirabbit antibodies as described in the previous paragraphs. In some experiments, the amount of mouse agrin bound was estimated by digital photomicrography using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image/).
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Acknowledgments |
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This work was supported by National Institutes of Health grant NS33213 to M.A. Smith.
Submitted: 6 January 2003
Revised: 5 May 2003
Accepted: 5 May 2003
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References |
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