Glia cell line-derived neurotrophic factorregulates the distribution of acetylcholine receptors in mouse primary skeletal muscle cells

Neuroscience 128 (2004) 497–509

GLIA CELL LINE-DERIVED NEUROTROPHIC FACTOR REGULATES THE DISTRIBUTION OF ACETYLCHOLINE RECEPTORS IN MOUSE PRIMARY SKELETAL MUSCLE CELLS L.-X. YANGa,b* AND P. G. NELSONa

Key words: neuromuscular junction, cell signaling, protein phosphorylation, synaptotrophic modulator, quantitative imaging, autocrine.

a

Section on Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bldg 49, Bethesda, MD 20892, USA b Department of Human Genetics, University of Pittsburgh, 633 Parran Hall, 130 Desoto Street, Pittsburgh, PA 15261-0001, USA

Substantial evidence has been obtained indicating that growth factors influence synapses in many parts of the nervous system. Synapse maintenance or modulation may be produced by target-derived neurotrophic materials acting on the neurons innervating those target cells (McAllister et al., 1999; Schuman, 1999). The neuromuscular junction (NMJ) has been used as a model for synaptic function and of the synaptic maturation process (Sanes and Lichtman, 1999). Valuable information has been provided by the NMJ where several trophic factors such as leukemia inhibitory factor, basic fibroblast growth factor, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5, influence developmental synaptic rearrangements (English and Schwartz, 1995; Gonzalez et al., 1999; Kwon et al., 1995; Kwon and Gurney, 1996; Lohof et al., 1993; Schuman, 1999). The mechanisms by which neurotrophic growth factors exert their actions on synapse formation and elimination remain elusive. Glial cell line-derived neurotrophic factor (GDNF) is well known for its trophic effects on the survival and differentiation of ventral midbrain dopaminergic neurons and various other neuronal populations including spinal motor neurons (Buj-Bello et al., 1995; Cacalano et al., 1998; Hashino et al., 2001; Henderson et al., 1994; Lin et al., 1993). GDNF is the first member of a newly established GDNF family ligands (GFLs) which include three other members: Neurturin (NRTN), Artemin (ARTN) and Persephin (PSPN; Baloh et al., 1998; Kotzbauer et al., 1996; Lin et al., 1993; Milbrandt et al., 1998). GFLs belong to the transforming growth factor-␤ superfamily, but the cell biology of the signaling for GFLs is complex. They utilize a two-component receptor system in which the transmembrane receptor tyrosine kinase c-Ret serves as a common signaling receptor with each of the different receptors of the GFR␣ family specifically preferred by the different trophic molecules (Airaksinen and Saarma, 2002; Nomoto et al., 1998; Takahashi, 2001). These receptors are members of glycosylphosphatidylinositol (GPI)-anchored co-receptors that bind ligands with high affinity and determine the specificity of the receptor complex: GFR␣1 for GDNF, GFR␣2 for NRTN, GFR␣3 for ARTN, and GFR␣4 for PSPN (Baloh et al., 2000; Cacalano et al., 1998; Masure et al., 2000; Nishino et al., 1999; Rossi et al., 1999). In vitro experi-

Abstract—It was recently reported that glia cell line-derived neurotrophic factor (GDNF) facilitates presynaptic axonal growth and neurotransmitter release at neuromuscular synapses. Little is known, however, whether GDNF can also act on the postsynaptic apparatus and its underlying mechanisms. Using biochemical cold blocking of existing membrane acetylcholine receptors (AchRs) and biotinylation of newly inserted receptors we demonstrate that GDNF increases the insertion of AChRs into the surface membrane of mouse primary cultured muscle cells and that this does not require protein synthesis. Quantitative data from doublelabel imaging indicate that GDNF induces a quick and substantial increase in AchR insertion as well as lateral movement into AchR aggregates, relative to a weak effect on reducing the loss of receptors from pre-existing AchR aggregates, which in contrast to the effect of PMA. These effects occur in both innervated and un-innervated muscles, and GDNF affects nerve-muscle co-cultures more than it affects muscle-only cultures. Neurturin, another member of GDNFfamily ligands has similar effects on AchRs as GDNF but the unrelated growth factor, EGF does not. Studies on protein phosphorylation and specific inhibitors of cell signal transduction indicate that GDNF function is mediated by receptor GFR␣1 and involves MAPK, cAMP/cAMP responsive elementbinding factor and Src kinase activities. GDNF may signal through c-Ret as well as NCAM-140 pathways since both the signaling receptors are expressed in the neuromuscular junction (NMJ). These data suggest that GDNF is an autocrine regulator of NMJ to promote the insertion and stabilization of postsynaptic AchRs. In vivo, GDNF may function as a synaptotrophic modulator for both pre- and postsynaptic differentiation to strengthen the functional and structural connections between nerve and muscle, and contribute to the synaptogenesis and plasticity of neuromuscular synapses. Published by Elsevier Ltd on behalf of IBRO. *Correspondence to: L.-X. Yang, Department of Human Genetics, University of Pittsburgh, 633 Parran Hall, 130 Desoto Street, Pittsburgh, PA 15261, USA. Tel: ⫹1-412-383-8733; fax: ⫹1-412-383-7844. E-mail address: [email protected] (L.-X. Yang). Abbreviations: AchR, acetylcholine receptor; AL-BTX, Alexa-Fluo-488conjugated ␣-bungarotoxin; ARTN, Artemin; ␣-BTX, ␣-bungarotoxin; CHX, cycloheximide; CNTF, ciliary neurotrophic factor; CREB, cAMP responsive element-binding factor; EGF, epidermal growth factor; GDNF, glia cell line-derived neurotrophic factor; GFLs, GDNF family ligands; GPI, glycosylphosphatidylinositol; MAPK, mitogen-activated protein kinase; NCAM, neural cell adhesion molecule; NMJ, neuromuscular junction; NRTN, Neurturin; PI-PLC, phosphatidylinositol– phospholipase; PKA, protein kinase A; PP, phosphoinositol phosphatase; PSPN, Persephin; RH-BTX, Rhodaminated ␣-bungarotoxin. 0306-4522/04$30.00⫹0.00 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2004.06.067

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ments showed that GDNF can crosstalk with GFR␣2 and GFR␣3, and GFR␣1 with NRTN and ARTN (Airaksinen and Saarma, 2002; Baloh et al., 1997, 1998; Trupp et al., 1998). In the GDNF system both GFR␣1 and c-Ret are necessary for GDNF signaling. Upon GDNF binding GFR␣1, c-Ret receptor tyrosine kinase is recruited and activated (Jing et al., 1996; Treanor et al., 1996). In situ hybridization studies showed that GFR␣s are more widely expressed in vivo than c-Ret, which suggests a possibility that GFR␣s may also signal in collaboration with novel transmembrane proteins (Masure et al., 2000; Trupp et al., 1997; Yu et al., 1998). Recent evidence from the c-Retdeficient cell lines and neurons indicates the alternative signaling mechanisms mediated by GFR␣1, while activating Src family tyrosine kinase (s) and subsequent phosphorylation of mitogen-activated protein kinase (MAPK), cAMP responsive element-binding factor (CREB; Poteryaev et al., 1999; Trupp et al., 1999). New study has identified the 140-kD transmembrane isoform of the neural cell adhesion molecule, NCAM (p140NCAM) as the alternative signaling receptor for the members of GFLs. Both GFR␣1 and NCAM-140 are required for GDNF to activate NCAM downstream signaling in immortalized neuronal precursors, in primary cultures of Schwann cell and in primary neurons which express high levels of NCAM and GFR␣1 but undetectable levels of RET (Paratcha et al., 2003). It was recently reported that in addition to its classic function, GDNF enhances spontaneous neurotransmitter release in amphibian neuron-myocyte co-culture and isolated neuromuscular preparations from mice (Ribchester et al., 1998). Transgenic mice with muscle-specific overexpression of GDNF have hyperinnervation of their muscle in newborn animals and delayed synapse elimination (Nguyen et al., 1998). Early postnatal s.c. injection of GDNF induces sustained multiple innervation by increasing in motor unit size and promoting axon branching at the NMJs (Keller-Peck et al., 2001). It is unclear whether, in addition to these significant positive effects on presynaptic differentiation, GDNF can act on the postsynaptic apparatus and, if so, what are the underlying molecular and cellular mechanisms. By definition, the process of synapse elimination must result in the disappearance of the presynaptic neurite, but experiments have indicated that an initiating step for the presynaptic removal is the diminution or disappearance of the postsynaptic acetylcholine receptors (AchRs) at synapses that are undergoing elimination (Balice-Gordon and Lichtman, 1993). Stabilization of the postsynaptic receptor could result in stabilization of the entire synapse and this has been demonstrated in vitro (Li et al., 2001, 2002). The GDNF receptors, GFR␣1 and Ret are found in the spinal motor neurons (Garces et al., 2000; Golden et al., 1999). Our previous studies revealed that all the isoforms of GFR␣1 (GFR␣1a-e) including the membrane-anchoring and soluble types are expressed in skeletal muscles (Yang and Kiuchi, 1998, 1999). We thought it would be worthwhile to test the hypothesis that in addition to its clear effect on the presynaptic neurites, GDNF might also have

an action on the postsynaptic receptors. If it did, it would be important for our understanding of the regulation of synapses by neurotrophic factors. To that end we have examined the action of GDNF on the metabolism and membrane distribution of the muscle AChRs in our established mouse primary culture systems, both in cultures containing only myotubes and in co-cultures of spinal cord neurons and muscle cells, and investigated the molecular and cellular mechanisms mediating the actions of GDNF in muscles. Portions of these results have appeared in abstract form (Yang et al., 2001; Yang and Nelson, 2002).

EXPERIMENTAL PROCEDURES Primary nerve-muscle culture preparations The 35-mm and 60-mm culture dishes were coated with laminin (1 mg/ml; Sigma, St. Louis, MO, USA). Thigh muscles from newborn mice were dissected, minced, and dissociated with the Papain Dissociation Kit (Worthington Bio-chemical Corporation). Animals were cared for in accordance with international guidelines for the humane treatment of laboratory animals. The number of animals used and their suffering were minimized in all experiments. The cell suspension was pre-plated onto a 100-mm dish for 30 min to allow fibroblasts to attach, then the nonadherent myocytes were plated with 10% FCS and 10% HS at 0.5⫻106 cells per 35-mm culture dish for imaging experiments and 1.5⫻106 cells per 60 mm culture dish for biochemical studies. The cultures were treated with 10⫺5 M cytosine ␤-D-arabino-furanoside (Sigma) for 36 h during the first week of plating to inhibit the growth of fibroblasts. The dissociated ventral horn neurons from embryonic day 13 mouse enriched for motor neurons by centrifugation through a 6.8% metrizamide gradient were added to the myotube cultures (Fig. 1). Neurons were treated with 13.4 mg/ml 5-fluoro-29-deoxyuridine (Hoffmann-La Roche) and 33.5 mg/ml uridine (Sigma) for 2 days after plating to inhibit growth of glia. The cultures were maintained in a 37 °C, 10% CO2 incubator and the fusion medium (MEM supplemented with 5% HS) was changed thrice a week. Experiments were carried out with 14-day-old cultures. For some experiments on muscles grown with nerve a three-compartment chamber (Lanuza et al., 2000) with muscle cells in the center chamber and GFP-neurons in the side chambers grown for about 3 weeks in vitro were used. Neurotrophic factors GDNF (rhGDNF 100 ng/ml; Promega) and NRTN (50 ng/ml; Chemicon), epidermal growth factor (EGF 50 ng/ml, Promega), and antibodies (antiGFR␣1 and anti-CNTFR; Transduction Laboratories) were added in the newly changed medium and control cells were in culture for the same length of time. To neutralize the biological activity of endogenous GDNF in the cultures 50 ng/ml anti-GDNF (Ab-2) monoclonal antibody (Oncogene, MA, USA) were used prior to experiments. Myotubes were pre-incubated with the inhibitors: 1 unit/ml phosphatidylinositol–phospholipase C (PI-PLC; Glyko), phosphoinositol phosphatase 2 (PP2; 1 ␮M), PP1/PP2 inactive analog PP3 (1 ␮M; Calbiochem), the cAMP blocker Rp8-Br-cAMPs (20 ␮M; Alexis), the MAP kinase inhibitor PD 98059 (25 ␮M; New England Bio Laboratories) or a diluted carrier control dimethyl sulfoxide (Sigma) for 45 min, then after washing out (three times with culture medium), GDNF was added to cultures with the inhibitors again.

Membrane biotinylation and quantitative immunoblotting analysis of AchR The cultured myotubes in 6-cm plates were initially incubated with 300 nM unlabeled ␣-bungarotoxin at 37 °C for 25 min to block the pre-existing membrane surface AchRs. After complete washout with the culture medium the cells were treated with reagents of

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Fig. 1. Microphotographs showing the fluorescence double-labeling method used to measure both the loss of AchR from labeled aggregates and the insertion of new receptors. (A) Brightfield view of primary myotubes. (B) Initial labeling of pre-existed AchRs with Alexa-␣BTX (AL1). (C) Second picture of initially Alexa-labeled AchR aggregate representing remained AchRs (AL2) at the end of the 4 h period. (D) Second labeling of newly inserted AchRs with RH-BTX (RH) in the previously defined area 4 h after the initial Alexa-labeling and photographed at the same time with AL2 by switching the filters. Other experiments showed that the initial Alexa-␣BTX labeling was with a saturating dose and that no RH-␣BTX labeling occurs immediately after the initial Alexa labeling.

interest for 1–12 h to allow the new receptors to insert into the membrane. The treatments were stopped by adding ice-cold PBS with Ca⫹⫹ and Mg⫹⫹ to wash and prevent receptor internalization. This was then followed by cell surface biotinylation at 4 °C for 20 min with 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce Chemical Company), or biotin-conjugated ␣-bungarotoxin (Molecular Probes, Eugene, OR, USA). The membrane biotinylation was stopped by washing with PBS once and changing to 20 mM glycine in PBS for a 15 min quench at room temperature and washing the cultures with PBS three times. The cells were lysed immediately with the M-PER mammalian protein extraction reagent (Pierce) plus 0.4% SDS, protease inhibitor cocktail and phosphatase inhibitor cocktail I and II (Sigma). To precipitate biotinylated proteins, supernatants of cell lysate were mixed with UltraLink immobilized Neutravidin beads (Pierce) and rotated for 2 h at 4 °C. After the NeutrAvidin Plus beads were washed five times with modified immunoprecipatation buffer (20 mM sodium phosphate pH 7.5, 500 mM NaCl, 0.4% SDS, 0.5% deoxycholic acid, 1% NP-40) and once with TBS, bound proteins were eluted in SDS-PAGE sample buffer containing 50 mM DTT at 90 °C for 15 min. Protein biotinylation was efficiently reversed by DTT in this procedure. Both total and biotinylated proteins were fractionated by SDS-PAGE on 4 –20% NuPAGE gradient Bis–Tris polyacrylamide electrophoresis gel (Invitrogen) and blotted to PVDF membranes (Millipore). Blots were probed with an anti-AchR ␣ antibody (BD Transduction Laboratories). Horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz, CA, USA) was used as secondary antibody to visualize the specific labeled protein bands by enhanced chemiluminescence (Amersham Phamacia Biotech, IL, USA). The membranes were scanned in a Storm 860 Imaging System and the scanned digital images were quantitated using

ImageQuant software (Molecular Dynamics). The amount of AchR surface expression was analyzed by determining the relative ratio of biotinylated AchR to total AchR. The biotinylated AchR signals were divided by the total AchR signals in each sample to obtain the ratio.

Fluorescence double-labeling of AchR and computerized image quantitation Pre-existing AchR aggregates on myotubes grown on 35-mm culture dishes were labeled and blocked with the Alexa-Fluo-488conjugated ␣-bungarotoxin (AL-BTX; at a saturating concentration of 300 nM, diluted in fusion medium) for 30 min followed by washing out of the unbound BTX. The images of fluorescently labeled AchR aggregates were collected using a NIKON Diaphot inverted microscope (60⫻ long working distance objective, N.A. 0.7) and a cooled CCD camera (Photometrics SenSys, Tucson, AZ, USA) connected to a Macintosh computer with IPLab software (BioVision Technologies, Inc.). An initial image (AchR-AL1) was acquired and the location of each aggregate was recorded. After incubation of the cultures with or without GDNF and the inhibitors for 1–12 h, the cultures were fixed with 2% paraformaldehyde and each imaged aggregate at several time points was re-located and the newly inserted AchRs in the previously defined areas of the cells were labeled with the Rhodaminated BTX (RH-BTX, 200 nM). A second AL-BTX picture (AchR-AL2) and the RH-BTX picture (AchR-RH) were taken at this time by switching the filters. See Fig. 1 for illustration of the technique. Intensity of aggregates was assessed by outlining the aggregate, copying that same “region of interest” to all subsequent images of the same aggregate and determining the intensity within the outlined regions.

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Background intensity measurements were collected for each image and subtracted from the aggregate intensity. Quantitations for the fluorescence intensities of AchRs were made to compare the intensity differences of AL1, AL2 and RH between GDNF-treated and un-treated cultures, including the comparisons of AL2/AL1 as the degradation rate and RH/AL1 as the insertion rate. The results of the quantitative analysis were expressed as mean⫾S.E. Significance was determined using Student’s t-test. In other experiments preexisting AchR aggregates were blocked with unlabeled ␣-BTX for 30 min (instead of AL-BTX as above) and then labeled with the Rhodaminated ␣-BTX (RH-BTX, 200 nM) after various periods of time. The newly inserted AchRs were then imaged and quantitated. All types of ␣-BTX were from Molecular Probes. In a typical single experiment, at least 26 aggregates were imaged on each culture plate of myotubes, with two plates for each experimental condition. Unless otherwise indicated at least three separate experiments were done for each condition. As in a previous study (Lanuza et al., 2000), we evaluated the effect of photobleaching and found it to be less than 5%; because of this small effect and our experimental design, the photobleaching did not affect our results.

Western blot analysis of protein expression and phosphorylation Primary cultured muscle cells in 6-cm plates were changed to serum-free media 16 h prior to incubation at 37 °C with GDNF for indicated time periods and immediately lysed with M-PER mammalian protein extraction reagent supplemented with 0.5% SDS and a mixture of protease inhibitors and phosphatase inhibitors (as above). Cleared total cell lysates were denatured with NuPAGE LDS sample buffer plus reducing agent (Invitrogen) at 72 °C for 15 min and resolved by SDS-PAGE on 4 –20% NuPAGE gradient Bis–Tris polyacrylamide electrophoresis gel and transferred to PVDF membranes. Blots were first probed with antiCREB (Ser133) and anti-phospho-Src (Tyr416) antibodies (Cell Signaling Tech). After stripping with 0.2 M glycine–HCl, pH 2.4 for 1 h at room temperature, the blots were re-probed with anti-CREB antibody and anti-c-Src antibody (Santa Cruz), respectively to demonstrate comparable amounts of CREB protein and pan Src protein in all the lanes. Western blot analysis of GFR␣1 expression in muscle was done using the muscle total cell lysates and probed with anti-GFR␣1 monoclonal antibody (BD Transduction Laboratories), and re-probed with Anti-actin (Ab-1) antibody (Calbiochem-Novabiochem Corporation, San Diego, CA, USA). Polyclonal antibody Anti-GFR␣1 (C-20), Anti-Ret antibodies, C-19 and C-20 were from Santa Cruz Biotechnology.

One-step RT-PCR and sequencing for Ret mRNA expression The total RNA from P9 in vivo and 13-day-cultured muscles and E14 ventral horn neurons was extracted using RNAqueous-4PCR Kit (Ambion). PCR analysis of Ret mRNA expression was performed with c-Ret sense primer, TGT ATG TAG ACC AGC CAG CT, and antisense primer, ACT ATG CAC AAA GCC TCC AG using Qiagen OneStep RT-PCR Kit (Qiagen, Inc). The 50 ␮l reaction mixture in 1⫻ of RT-PCR buffer contained 1.5 ␮g of the extracted total RNA, 0.5 ␮M each of the sense and antisense primer, 2 ␮l of the enzyme mix (a mixture of heterodimeric recombinant reverse transcriptases Omniscript, Sensiscript and HotStar TaqDNA polymerase), 400 ␮M of each dNTPs (dATP, dCTP, dGTP and either dTTP), 1⫻ Q solution (to adjust the melting temperature of primers). The reaction was subjected to a precycle condition consisting of 30 min at 50 °C (for reverse transcription), 15 min at 95 °C and the cycling conditions were 94 °C for 45 s, 57 °C for 50 s and 72 °C for 1 min for 35 cycles followed by 72 °C for 8 min. Amplified DNA was separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The

amount of total RNA for each sample was quantitated with the UV spectrophotometer and verified by the same one-step RT-PCR using primers for the housekeeping gene with the same amplifications for all the samples. The identity of each expected band was confirmed by cloning into pCR 4-TOPO vector (Invitrogen) and nucleotide sequence analysis.

RESULTS GDNF increases the appearance of AChR into the surface membrane of cultured myotubes To establish whether GDNF would have any effect on the metabolism or turnover of AchR and receptor movement into the surface membrane of muscle we performed a biochemistry method of membrane biotinylation in which the pre-existing AchRs were blocked with unconjugated ␣-BTX followed by a treatment period and labeling of newly inserted membrane receptors with biotinylated ␣-BTX, then the membrane receptors were immunoprecipitated and subjected to quantitative blotting analysis. The time course of these experiments was such that no difference between treated and untreated cultures was evident at 1.5 h, because the level of newly inserted AchR is still very low at this time (data not shown). An increased insertion of membrane AChR in GDNF-treated cultures, however, could be demonstrated after 4 and 8 h, as shown in Fig. 2A (GDNF vs. control at 4 h), a robust increase in AchR amount in the myotube surface membrane (Membr AchR) but no change in the total AchR (Total AchR). To determine whether this increase in surface AchR required protein synthesis or not, we compared the GDNF effect in the presence and absence of protein synthesis blocker, cycloheximide (CHX), as shown in Fig. 2A. There was no difference in receptor insertion in these two conditions (compare GDNF with CHX, the latter representing co-treatment with GDNF and CHX). Since the total AchR of the muscle (including both surface and intracellular components) was relatively stable and not affected by GDNF action, it was used as a normalizing factor for the quantitations of AchRs in the surface membrane of myotubes. The quantitative results from four experiments are shown in Fig. 2B. GDNF affects nerve-muscle co-cultures more than it affects muscle-only cultures We also investigated whether the cell response to GDNF might be affected by the co-culture of myotubes with spinal neurons since neuronal activity usually has some influence on AchRs. When the insertion of AChR was examined in cultures containing either myotubes alone or myotubes co-cultured with ventral spinal cord neurons, the response to GDNF was significantly larger in the co-cultures than in the myotube-only cultures (Fig. 3A). Quantitative data from five experiments for both innervated and noninnervated muscle are shown in Fig. 3B. The differences in membrane insertion of AchR metabolism between treated and untreated cultures (both with muscle alone or in co-cultures) are quite significant (P⬍0.01 and P⬍0.001, respectively) and that between treated/innervated and treated/uninnervated cultures is also statistically significant (P⬍0.05). With the same method, however, the other growth factor

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Fig. 2. Treatment with GDNF increases the appearance of AchR in the co-cultured myotube surface membrane. Western blots of AchR (49 kDa) are shown from untreated myotube-neuron co-cultures and sister cultures treated with GDNF. Total cell AchRs are shown as well as the membrane AchRs recovered 4 h after cold block of the existing AchRs on the muscle cells and by immunoprecipitation of the biotinylated surface membrane receptors. Pre-treatment with CHX does not inhibit the GDNF effect. (A) Representative blots of control, GDNF treated and GDNF plus CHX for membrane and total AchRs. (B) Quantitative results from four experiments. * Different from control, P⬍0.01. In this and subsequent figures, the error bars represent the S.E.M.

EGF has no such effect on AchRs as GDNF does (data not shown). AchR concentration is increased in the receptor aggregates by GDNF Total insertion of the AchR is an important determinant of receptor function, but the spatial organization of the receptor is also crucial. In particular, the accumulation of the receptors in high-density aggregates is a prominent aspect of receptor biology. We used fluorescence imaging techniques to examine the question of possible involvement of GDNF in the spatial organization of AchR. Our initial experiment was to accurately measure both the average brightness of the labeled receptor aggregates and the average area of the aggregates that were above threshold intensity. The fluorescent brightness or intensity represents the number or concentration of the AchRs in the aggregates. In order to be sensitive to the rate of insertion of AchRs, all the receptors were initially blocked with unlabelled ␣-BTX before treatment. Four hours later we labeled the myotubes with Rhodamine-labeled BTX and compared GDNF-treated cultures with control cultures with regard to the amount of labeled BTX that could be measured in AChR aggregates. GDNF-treated cultures exhib-

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Fig. 3. GDNF affects nerve-muscle co-cultures more than it affects muscle-only cultures. Western blot analysis of experiments such as that shown in Fig. 2 for cultures of myotubes alone or for myotubeneuron co-cultures. (A) Representative blots showing membrane AchRs (upper) and total cellular (lower) AchRs in muscle alone and in co-cultures as indicated. (B) Quantitative data for five experiments as labeled. * P⬍0.01; ** P⬍0.001.

ited a significantly higher intensity of AchR labeling than did the control cultures but the area of aggregates in the treated cultures was not significantly larger than in the control cultures. Quantitative data for three experiments of this kind are shown in Fig. 4. This result confirmed the biochemical experiments shown in the previous figures and indicates that the balance of AChR insertion and degradation is shifted toward more insertion into the highdensity receptor aggregates. As noted using biochemical methods above, the GDNF effect on the AchRs measured by imaging methods was also not affected by the protein synthesis inhibitor, CHX (data not shown). GDNF affects AchR insertion more than degradation in aggregates In the foregoing, we sought to test further the assumption that the increase in AchR in the surface membrane reflects an increased rate of insertion of the receptor. In order to study simultaneously and more directly both the insertion of the receptors into aggregates (their appearance in the aggregates) and the loss of receptors (disappearance from the aggregates) we developed a fluorescence double-labeling method as described in the Experimental Procedures (Fig. 1). A saturating dose of Alexa labeled ␣-BTX was used to fully block and label the receptors and this initial labeling (Fig. 1B) was quantitated as

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Fig. 4. The intensity of labeling of AchR aggregates is increased by GDNF treatment but the area of the aggregates is not. Quantitative imaging of initially blocked all the AchRs in aggregates with unlabelled ␣-BTX followed by 4 h incubation with or without GDNF treatment and then labeled the AchR aggregates in myotubes with RH-BTX. These data were from nerve-muscle co-cultures including plates of threecompartment chambers. The normal and GDNF-treated data are from paired four plates in each of three separate experiments. The S.E.M. bars are for variation in the average intensity and area obtained from each of the three experiments.

AL1. After various periods of incubation or treatment (from 1 to 12 h) the remaining amount of the initially labeled receptors was then measured in the same aggregate and quantitated as AL2 (Fig. 1C). At this time point, a second labeling with RH-BTX was measured as RH (Fig. 1D), which gave an index of how much receptor had been inserted into the aggregate during the time period. There was no labeling with the RH-␣-BTX immediately after the Alexa labeling since saturating doses of the Alexa ␣-BTX were used in the initial labeling (data not shown). Fluorescence intensities involving untreated control cultures are compared with that from GDNF-treated cultures. The results of such experiments are shown in Fig. 5 with the intensity of initial labeling (AL1), the amount of the initial label remaining after 90 min (AL2), and the amount of newly inserted receptor (RH). There was no difference between control and GDNF treated cultures for AL1 since this measure reflects only the initial block of labeling prior to treatment, and the random selection of aggregates would not be expected to produce any difference in receptor concentration or number. There is not a significant effect on receptor loss (indicated by AL2) at this 1.5 h time point (but see below). There is a highly significant increase in the insertion of AchR as indicated by the higher intensity of RH in the GDNF treated cultures. The time course of changes in AL2 and RH from 0 to 12 h in one representative experiment is shown in Fig. 6A. A modest decrease of the Alexa ␣-BTX occurs with time in

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Fig. 5. Receptor appearance in the aggregates is increased but loss is not affected by GDNF after 90 min of treatment. Quantitative doublelabel imaging shows the effect of GDNF on Alexa labeling after 90 min (AL2) and RH-BTX at the 1.5 h time point. As expected there was no detectable difference between the two populations of aggregates with respect to AL1 (initial labeling). Note no effect of GDNF on AL2, but highly significant effect of GDNF on RH-labeling (newly inserted AchRs). ** P⬍0.001, data from three experiments for cultures of myotubes alone.

control preparations, as the initially labeled AChR is lost from the aggregate. A significant amount of RH-␣-BTX labeling occurs at various times after the initial labeling as new receptor is inserted into the aggregates. The difference between RH measured in control and GDNF treated cultures increases for the first 4 h and then stays relatively constant out to 12 h. A slight decrease of receptor loss (i.e. the diminution of loss produced by GDNF) is shown by the higher level of AL2 in the treated cultures of 8 h. This effect on loss is significant, but the effect of GDNF on the insertion of AChR into the membrane is quantitatively much more substantial. The same experiments were repeated twice with the similar results. These data suggest that the major action of GDNF is primarily on the process of receptor insertion, especially with relatively little effect on receptor degradation. The specificity of these results for GDNF treatment is shown by comparison of Fig. 6A with data from another treatment, namely the PKC activator, PMA (Fig. 6B). This agent produced a strong effect (an increase) on loss of AchR from the membrane but has no significant effect on receptor insertion, in contrast to the effect of GDNF. Further evidence of the specificity is shown by experiments with different trophic factors. Both GDNF and NRTN (another member of GFLs) produce an increase of AchR insertion and lateral movement into the AchR aggregates in the myotube membrane, while the unrelated growth factor, EGF does not (Fig. 7).

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12

0.5

Time (hours)

B

560

Ctr-RH

Fluorescence Intensity of AchR

480

*

400

PMA-RH Ctr-AL

*

320

0

Ctr

EGF

PMA-AL

*

240

*p < 0.01

160 80 0 0

1

2

3

4

5

6

7

8

Fig. 7. GDNF and its family member NRTN affect the AchR but EGF does not. Quantitative double-label imaging experiments showing different effects after 2 h of treatment with different growth factors. Ctr, control; GDNF, NRTN and EGF. The data are plotted as AL2/AL1 for Degradation and RH/AL1 for Insertion. * Difference between GDNF treated and control preparations from muscle alone is significant at P⬍0.001.

9

Time (hours)

Fig. 6. Time course of AchR double-labeling and the effect of GDNF contrasting to that of PMA treatment. (A) Compared with the profound effect of GDNF on AchR insertion at early time point relative to a minor effect of GDNF on receptor loss after 8 h treatment: difference between control and GDNF treated myotubes alone preparations is significant at P⬍0.01 and P⬍0.05, respectively. These data are from a representative experiment, which was repeated twice with similar results. Each time point represents a separate set of myotubes from different plates with each aggregate being imaged at t⫽0 and the indicated time point. (B) Experiments like that in (A) but with PMA treatment to show the contrasting effect to that seen with GDNF treatment. The standard deviations of the data are not shown for clarity in the figure.

GDNF receptors are expressed in the muscles as well as motoneurons GFR␣1 is the specific binding receptor for GDNF; therefore, a prerequisite for the action of GDNF on muscle is the expression of the receptor GFR␣1 in muscle. Western blot analysis shown in Fig. 8A demonstrates such expressions in cultured and in vivo muscles as well as in neurons but not in the spleen. Higher expression level of GFR␣1 protein could be detected in the P nine in vivo muscle than in the 13-day cultured myotubes. The specificities of antibodies were tested by immunoprecipitation and Western blot analysis (data not shown). In many culture systems the transmembrane receptor c-Ret works with GFR␣1 to mediate the actions of GDNF. In muscle Ret is present in very low levels and we were unable to show the evident presence of this protein by Western blot. With RT-PCR, however, we can clearly demonstrate the mRNA expression of Ret in muscle and spinal

cord, a single 615-bp band of cDNA as expected (Fig. 8B) from both in vivo and in vitro tissues but not in the negative control lane (without template). We have cloned the PCR products into TA cloning vector followed by nucleotide sequence analysis, which confirmed that the cDNA sequence is identical to that of mouse Ret gene in the proteincoding region (data not shown). To test the specificity of GDNF signaling and its receptor involvement, the quantitative imaging of AchR doublelabel was performed as an assay for the possible cell signaling pathways and the roles of biological coupling components mediating GDNF action (Table 1). When cultures were treated with anti-GFR␣1 antibody which blocks the receptor function, the action of GDNF on AchRs was abolished. GDNF effect was not blocked by another antireceptor antibody that against the CNTF receptor ␣. GDNF works on the AchRs by activating MAPK, cAMP/CREB and Src tyrosine kinase After binding to its two-component receptor complex, GDNF activates several downstream intracellular pathways, including the MAPK and CREB. As shown in Table 1, the MAPK blocker PD 98059 prevented the GDNF elicited increase in membrane AchRs. The cAMP blocker Rp-cAMP also inhibited GDNF action on AchR membrane insertion. Another cell biologic pathway that has been established as mediating some of GDNF actions involves the tyrosine kinase, Src. A substantial blocking on the GDNFinduced insertion Rate of AChRs was obtained by treatment with the Src kinase inhibitor, PP2. No effect was seen with the PP2 inactive analog PP3.

504

Table 1. Specificity of the effects of GDNF and its inhibitors on AchR

1

3

2

4 GFRα1

Actin

B

1

2

3

4

5

RET

Fig. 8. GDNF receptors are expressed in muscles. (A) Western blot analysis showing GFR␣1 protein (52 kDa) is expressed in muscle cells both in vivo and in vitro. Lane 1, sample from in vivo P9 muscles; lane 2, sample from P13 cultured muscles; lane 3, same amount of protein as other lanes but from spleen was loaded; lane 4, from E-14 day spinal cord ventral horn neurons. The lower panel shows a re-probing of the same filter with anti-Actin antibody and demonstrates comparable amount of Actin protein (42 kDa) in all the lanes. (B) The Ret gene is transcribed in muscle cells. One-step RT-PCR was performed with primers for Ret with a single 615-bp band of cDNA as expected. Lane 1 was with material from in vivo P9 muscle, lane 2 was GDNF treated P13 cultured myotubes, lane 3 was from untreated P13 cultured myotubes, lane 4 was a negative control (without template) and lane 5 was from E-14 day ventral horn neurons. The same amount of total RNA was used as the template for each of the amplifications. The bands were cut out and PCR products were cloned and sequenced; the cDNA sequence was identical to the mouse Ret gene.

As noted above, the effect of GDNF on loss of receptor is less pronounced than its effect on insertion of the receptor and the effect on loss at 2 h was not significant, so the effects of the inhibitors were undetectable at this early time point. At 8 h, however, there was a significant effect of GDNF on receptor loss (P⬍0.05), and this effect was also blocked by the anti-GFR␣1 antibody, the MAP kinase blocker, the cAMP antagonist and the Src kinase inhibitor (Table 1). These results using the morphological analysis of AchR appearance in surface aggregates were confirmed by parallel experiments using the biochemical methods described above. As shown in Fig. 9, the antibody against GFR␣1 blocked GDNF action on the AchR as did RpcAMP and the Src kinase blocker PP2, while PP3 had no effect. In addition, GFR␣1 is anchored to the cell membrane through a GPI linkage that is important for the classic receptor function. This linkage is broken by the phospholipase, PI-PLC, which removes GPI-linked proteins from the cell membrane. This enzyme also significantly diminished the effect of GDNF on the AchRs although not completely blocked (Fig. 9). If GDNF worked through Src and cAMP/CREB as suggested by the inhibitor experiments, an expected result would be phosphorylations of CREB and the Src tyrosine

2h Control GDNF GDNF⫹inhibitors: Anti-GFR␣-1 Anti-CNTFR␣ Rp-cAMP PD 98059 PP2 PP3 8h Control GDNF GDNF⫹inhibitors: Anti-GFR␣-1 Rp-cAMP PD 98059 PP2 PP3

Degradation rate1

Insertion rate1

0.79⫾0.04 0.84⫾0.05

0.90⫾0.04 1.33⫾0.05a

0.78⫾0.03 0.85⫾0.05 0.77⫾0.03 0.79⫾0.03 0.78⫾0.04 0.84⫾0.05

0.87⫾0.06 1.29⫾0.05a 0.89⫾0.05 0.83⫾0.05 0.84⫾0.06 1.27⫾0.05a

0.59⫾0.04 0.68⫾0.05b

1.37⫾0.07 1.78⫾0.09a

0.56⫾0.04 0.52⫾0.03 0.51⫾0.03 0.57⫾0.04 0.67⫾0.06b

1.35⫾0.06 1.34⫾0.06 1.34⫾0.07 1.36⫾0.08 1.76⫾0.09a

1

Degradation rate⫽AL2/AL1; insertion rate⫽RH/AL1. P⬍0.001; b P⬍0.05, significantly different from control. a

A

Ctr

RAb

PIP

RpA

PP2

PP3

Membr

Total

B

0.8

Membr/Total (Arbitrary Unit)

A

L. X. Yang and P. G. Nelson / Neuroscience 128 (2004) 497–509

0.6

* *

*

*

0.4

0.2

0

Fig. 9. GDNF effects are mediated through the receptor GFR␣1 and involve cAMP and Src kinase. (A) Representative Western blots showing membrane AchRs (upper) and total cellular (lower) AchRs (49 kDa). (B) Quantitative data from 5 experiments with the cultures of muscle alone, comparing effect of GDNF (lane 1, as control) and GDNF plus a number of inhibitors: RAb is the anti-GFR␣1 antibody; PIP is the phospholipase, PI-PLC that cuts the phospholipid anchor for the receptor; RpA is Rp-cAMP, an antagonist of cAMP; PP2 is the Src kinase family blocker; PP3 is the inactive analog of PP2. * P⬍0.01.

L. X. Yang and P. G. Nelson / Neuroscience 128 (2004) 497–509

A

0

5

15

30

60

GDNF (min) Phospho-CREB (Ser133) CREB

B

0

5

15

30

60

GDNF (min) Phospho-Src (Tyr416) c-Src

Fig. 10. Time-course of GDNF-induced phosphorylation of CREB and activation of Src tyrosine kinase in primary muscle cultures. (A) Rapid phosphorylation of CREB in Ser-133 demonstrated by immunoblot of total cell lysates with a specific anti-phospho-CREB antibody (upper panel). The lower panel shows a re-probing of the same filter with anti-CREB antibody and demonstrates comparable amounts of CREB protein (43 kDa) in all the lanes. (B) Tyrosine kinase phosphorylation of Src in Tyr-416 (upper panel). The lower panel shows a re-probing of the same filter with c-Src antibody and demonstrates comparable amounts of pan Src protein (60 kDa) in all the lanes. Phosphorylation peaks a little earlier for CREB (15 min) than for Src (30 min). The results shown are representative of three independent experiments.

kinase itself, since Src activation involves its phosphorylation. We have examined the effects of GDNF action on CREB and Src phosphorylations by Western blot analysis with the corresponding phospho-specific antibodies. The time-course of GDNF-induced phosphorylations of CREB in Ser-133 and Src kinase in Tyr-416 is shown in Fig. 10. Phosphorylations of both of these molecules increased more than threefold as compared with the controls (upper panels in A and B). Phosphorylation peaks a little earlier for CREB (15 min) than for Src (30 min). The stripping and re-probing with anti-CREB and pan Src antibody show the equal protein loading in each lane (lower panels in A and B). As expected, these phosphorylations are also inhibited by anti-GFR␣1 antibody (data not shown).

DISCUSSION The results reported here show that certain of the components of the system involved with GDNF action occur in skeletal muscle and that this system can regulate significantly the appearance of the AchR in myotube surface membrane without significantly affecting receptor synthesis and in the presence of a protein synthesis inhibitor, suggesting that GDNF has its effect on the surface AchR level by means other than a change in synthesis of the receptors. Both GDNF and NRTN but not EGF can produce a significant increase in AchR density. We saw this increase of receptor in membrane in the early period using both biochemical measurements (Western blot analysis of newly inserted receptors) or imaging techniques (which quantify the receptor aggregates on the myotube surface). We were interested whether the GDNF effect might be affected by the co-culture of myotubes with spinal neurons and the biochemical method allowed us compare this change in different conditions at once. In addition to the influence of neuronal activity on AchRs (compare the AchR

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levels of muscle only and co-culture), the effect of GDNF is significantly increased in nerve-muscle co-cultures. It may be that this “extra effect” is mediated through the neurons. Schwann cell-derived GDNF is taken up by motor neurons and transported anterogradely along the axons for release from terminals. It can escape the degradative pathway upon internalization and are cycled for future uses (Rind and von Bartheld, 2002; Russell et al., 2000; von Bartheld et al., 2001). Alternatively, the neurons may have some modulating effect on the muscle such that the muscle’s response to GDNF is increased, or perhaps via Agrin or other agents released from the nerve under GDNF treatment acting on the muscle cells. In any event, our results suggest that GDNF may be mediating synaptic plasticity in both a retrograde fashion (released from muscle to affect the presynaptic nerve terminal’s function) and in an autocrine mode to maintain a high concentration of AchR at the synapse. The double-label imaging experiments allowed us to focus on the high-density AchR aggregates and to monitor the AchR turnover and lateral movement, thereby precisely measuring both the loss of pre-existing receptors and the incorporation of new receptors in the same aggregates in the surface membrane at once. The data of Fig. 6A strongly suggest that the major effect of GDNF is on the quick insertion of receptor into the surface membrane and that it also has a significantly lesser effect on the stability of AchR, i.e. on the rate of loss from the membrane later. There is some decrease in the rate of receptor loss under GDNF treatment, but the insertion rate is affected more. We base this conclusion on the following argument. The amount of receptor in aggregates in control cultures is quite stable over the time course of the experiments shown in Fig. 6A, so the decrease of Alexa-label signal at 12 h is equivalent to the increase in Rhodamine signal at the same time. Thus 291 Alexa units are equivalent to 467 Rhodamine units. The decreased loss of Alexa label is equivalent to some 39 units of fluorescence intensity at 4 h while the increased Rhodamine signal is 134 units. Normalizing for the difference in signals generated by the two labels (467/291 Rhodamine units per Alexa unit) shows that the increase in the rate of receptor insertion measured by the Rhodamine staining is more than twice the decreased loss as measured by the Alexa staining. The high specificity of the effects of different phosphorylation reactions (i.e. the different kinases) on AchR movements is demonstrated by the dramatic differences between the effects of GDNF and PMA. PKC activation by the phorbol ester results in an effect exclusively of increasing AchR loss from the surface membrane, as shown in previous work (Lanuza et al., 2000). GDNF, in contrast, works primarily by increasing insertion of the receptor into the surface membrane. To elucidate the molecular mechanisms mediating the actions of GDNF in muscles, the existence of receptors for the GDNF is essential for the trophic factor to play a direct functional role in regulating muscle properties. A key requirement for the action of GDNF on muscle is the expression of GFR␣1 in muscle since it is needed for both c-Ret

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and NCAM-140 signaling pathways. Our previous studies have demonstrated that GFR␣1 mRNA is well expressed in mouse muscle (Yang and Kiuchi, 1998, 1999; and our unpublished observations). Here we demonstrate that GFR␣1 protein is evidently expressed in the muscles as well as neurons (Fig. 8A). As noted above, the GFR␣1 protein expression in vivo P9 is more than that in the13day cultured myotubes, because the younger in vivo muscles contain the soluble isoforms, GFR␣1d and GFR␣1e, while they may be washed out from the cultured myotubes. Both GFR␣1d and GFR␣1e have the hydrophilic Cterminals lacking the hydrophobic domain for glycosylphosphatidylinositol (GPI) attachment to the cell membranes (Yang and Kiuchi, 1999). Consistent with the findings above, GDNF action was completely blocked by treatment with anti-GFR␣1, the function-blocking antibody and significantly reduced by the phospholipase, PI-PLC in the biochemical experiments. PI-PLC has been shown to down-regulate the action of GDNF in a number of systems (Baloh et al., 1997; Jing et al., 1996; Nozaki et al., 1998). Taken together, it is concluded that GDNF works on the AchRs through its conventional receptor-binding subunit GFR␣1. Similarly GFR␣2 also expresses in skeletal muscle and neurons during development (Buj-Bello et al., 1997; Klein et al., 1997) for the NRTN actions on AchR regulation. In addition, the two GFLs could have some crosstalk with the two binding receptors. Previous study has failed to find Ret in human skeletal muscle with in situ hybridyzation (Kami et al., 1999), but RT-PCR results showed low expression level of Ret mRNA in rat muscle (Russell et al., 2000). Our one-step RT-PCR data confirmed by sequence analysis are compelling evidence that Ret is expressed in mouse muscle cells, both in vitro and in vivo. While myotube cultures may contain occasional fibroblasts and Schwann cells, these cells do not express Ret (Paratcha et al., 2003) and thus would not contribute to the RT-PCR amplification. Ret is expressed in the postsynaptic muscle fiber and not, as might be expected, exclusively in presynaptic motor nerve terminals. Ret may play certain roles in synaptic maintenance at the NMJ in some period of development. Furthermore, the newly identified alternative signaling receptor for GFLs, the transmembrane NCAM isoform, p140NCAM has been previously well documented expressing in muscles (Covault et al., 1986; Figarella-Branger et al., 1990; Moore et al., 1987; Rieger et al., 1985; Rutishauser et al., 1983). NCAM adhesion is essential for the in vitro establishment of physical associations between nerve and muscle, which may be an important early step in synaptogenesis (Rutishauser et al., 1983). We thus suggest that GFR␣1 mediated postsynaptic signaling by GDNF is most likely through p140NCAM because this NCAM-140 isoform not only exists specifically in mouse skeletal muscle but also co-localizes well with AchR at nerve-muscle contacts, and can be regulated during development, denervation and regeneration of skeletal muscle (Rieger et al., 1985). Intriguingly, some biological functions including axonal growth, synaptogenesis and synaptic plasticity previously ascribed to homophilic NCAM

interactions could actually be mediated by GDNF since Src, CREB and MAPK activities are also involved in the NCAM signaling pathways as that involved downstream of Ret signaling. Further studies (including on gene knockout mice) would explain the versatile molecular and cellular mechanisms in detail and the relationships between the Ret and NCAM signaling pathways at the NMJ. CREB is one of the targets of GDNF action, and phosphorylation of CREB involves cAMP and cAMP-dependent protein kinase (protein kinase A or PKA) action (Airaksinen and Saarma, 2002). Similarly, the cAMP blocker Rp-cAMP reduces GDNF effects. Thus GDNF action may require, at least in part, cAMP and PKA activation (Fukuda et al., 2002). The CREB phosphorylation suggests that GDNF may have an effect on gene transcription, but its action on AchR does not require altered protein synthesis; rather its major action is on the insertion and stability of pre-existing receptors in the aggregate. It has been reported that GDNF signal transduction pathways, including NCAM-140, involve MAP kinase in dopaminergic neurons and cortical neurons (Nicole et al., 2001; Worby et al., 1996). Here our imaging experiments with the MAPK blocker PD 98059 demonstrate that MAP kinase activity is also necessary for GDNF action on the AchRs. GDNF action also has a component dependent on Src kinase activity since the Src kinase blocker, PP2, prevents the trophin’s effect on the AchRs. Src kinase involvement in GDNF action has been seen in other systems (Encinas et al., 2001; Trupp et al., 1999). Indeed, Src kinase has been shown to mediate the actions of Agrin and MuSK, major determinants of NMJ formation (Mohamed et al., 2001). By immunoprecipitation we observed that Src tyrosine kinase binds to the AchR of skeletal muscle (data not shown), which suggests that GDNF may also interact with this powerful receptor regulating system. Our result showing that GDNF action increases Src phosphorylation is consistent with this interpretation. The evidences above suggest that the effect of GDNF on both insertion and loss of AchR utilize similar cell biological pathways. The AchR is known to be a target for several kinases with different receptor subunits and different amino acid residues being phosphorylated by the different kinases (Swope et al., 1999). Each phosphorylation event has a distinct effect on receptor stability or movement into or out of the muscle cell membrane. The action of PKA for instance, is known to stabilize the receptor in myotube membrane (Li et al., 2001). Activation of the Src tyrosine kinase (with other tyrosine kinases affected by Agrin and MuSK activity) is involved in the lateral movement of the receptor into aggregates (Mittaud et al., 2001; Mohamed et al., 2001). PKC is a serine kinase producing an increased rate of loss of AchR from aggregates (Lanuza et al., 2000; Li et al., 2004). Our results showing that GDNF action involves activation of both PKA and a tyrosine kinase suggest the mechanisms that account for the GDNF effects of slowing loss (perhaps due to PKA action) and increasing insertion and movement (attributable to Src tyrosine kinase activity) of AchRs from and into, respectively, aggregates on the myotube surface membrane.

L. X. Yang and P. G. Nelson / Neuroscience 128 (2004) 497–509

As noted, the effect of GDNF on synaptic transmission has generally been considered to have a pre-synaptic locus. Striking effects of GDNF on transmitter output, increase in the number and the size of synaptic vesicle clustering have been demonstrated (Liou et al., 1997; Ribchester et al., 1998; Wang et al., 2002). Those experiments also revealed a small but significant effect on spontaneous end plate potential amplitude, implying a probable postsynaptic effect on receptor density at the synapse (Wang et al., 2002). The latter experiments involved very young (1–3 days old) Xenopus cultures. Our results demonstrate that even after only 90 min, a significant increase in AchR density can be produced by GDNF and NRTN, and even in the absence of nerve cells; therefore, a direct effect of GDNF on the muscle cells must be involved. Comparable postsynaptically mediated effects on synaptic transmission due to an autocrine action of GDNF can be anticipated since both GDNF and its receptors are expressed in the muscles (Nagano and Suzuki, 2003). We thus conclude that GDNF regulates not only presynaptic differentiation and neurotransmitter release but also the postsynaptic insertion, distribution and stabilization of AchRs. As an endogenous growth factor, GDNF may function as a synaptotrophic modulator in vivo. This would be expected to strengthen the functional and structural connections and could contribute to the plasticity of neuromuscular synapses. Acknowledgments—The authors thank Ms Veronica Dunlap for helpful tissue dissection assistance.

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(Accepted 28 June 2004) (Available online 9 September 2004)