Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells expressing ErbB4

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 26 (2004) 271 – 281

Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells $ expressing ErbB4 Mendell Rimer, a,b,* Anne L. Prieto, c,1 Janet L. Weber, c Cesare Colasante, b,2 Olga Ponomareva, b Larry Fromm, a,3 Markus H. Schwab, c,4 Cary Lai, c and Steven J. Burden a a

Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, NYU Medical School, New York, NY 10016, USA Section of Neurobiology, Institute for Cell and Molecular Biology, and Institute for Neuroscience, University of Texas at Austin, Austin, TX 78712, USA c Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, USA b

Received 6 November 2003; revised 29 January 2004; accepted 11 February 2004 Available online 27 March 2004

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin2 is a candidate for a signal that regulates synaptic differentiation. D 2004 Elsevier Inc. All rights reserved.

Introduction The formation and maintenance of synapses depends upon an exchange of signals between presynaptic and postsynaptic cells. Three signaling pathways regulate acetylcholine receptor (AChR) expression at the neuromuscular junction (NMJ). First, neuronal

$ Supplementary data associated with article can be found, in the online version, at doi:10.1016/S1044-7431(04)00032-6. * Corresponding author. Section of Neurobiology, The University of Texas at Austin, 1 University Station C0920, Austin, TX 78712-0248. Fax: +1-512-471-9651. E-mail address: [email protected] (M. Rimer). 1 Current address: Department of Biology, Indiana University, Jordan Hall Room 341, Bloomington, IN 47405, USA. 2 Permanent address: Laboratorio de Fisiologı´a de la Conducta, Facultad de Medicina, Universidad de Los Andes, Me´rida 5101, Venezuela. 3 Current address: Ball State University, 221 N. Celia Avenue, Muncie, IN 47303-4609, USA. 4 Current address: Max-Planck-Institut fu¨r experimentelle Medizin, Hermann-Rein-Str. 3, 37075 Goettingen, Germany. Available online on ScienceDirect (www.sciencedirect.com.)

1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.02.002

isoforms of Agrin stimulate the muscle-specific receptor tyrosine kinase, MuSK, leading to the clustering of a variety of musclederived proteins, including AChRs, to synaptic sites (Cohen et al., 1997; Glass et al., 1996; Jones et al., 1997; McMahan, 1990). Genetic studies demonstrate the critical roles for Agrin and MuSK in synapse formation, as mice lacking either Agrin or MuSK fail to form neuromuscular synapses and die at birth (DeChiara et al., 1996; Gautam et al., 1996). Second, AChR genes are transcribed preferentially in myofiber nuclei that are positioned near the synaptic site causing AChR mRNA to accumulate at synaptic sites. The signals responsible for synapse-specific transcription remain elusive, but Neuregulin-1 (Nrg-1) (Falls et al., 1993) is a favored candidate (for reviews, see Fischbach and Rosen, 1997; Schaeffer et al., 2001). Third, ACh-evoked action potentials, which propagate along the entire myofiber, suppress AChR transcription throughout the myofiber. Consistent with the idea that Nrg-1 is a signal for synapsespecific transcription, adult mice that are heterozygous for a nrg-1 allele, lacking the immunoglobulin-like domain, are mildly dystrophic and have fewer AChRs at their NMJs (Sandrock et al., 1997). Synapse-specific transcription is qualitatively normal, however, in newborn mice lacking motor neuron-derived Nrg-1 (Yang et al., 2001), raising the possibility that muscle-derived Nrg-1, or signals other than Nrg-1, regulate synapse-specific transcription. Three nrg-1-related genes, nrg-2, nrg-3, and nrg-4, have been identified (Busfield et al., 1997; Carraway et al., 1997; Chang et al., 1997; Harari et al., 1999; Higashiyama et al., 1997; Zhang et al., 1997). Although their expression patterns have not been studied in detail, nrg-2 and nrg-3 are expressed predominantly in the nervous system, with notable expression in the cerebellum and hippocampus. Like Nrg-1, these Nrgs bind and activate ErbB3 and ErbB4, receptor tyrosine kinases that can dimerize with ErbB2. The different Nrgs, however, display preferences for the different ErbB receptors, as Nrg-1 binds equivalently to ErbB3 and ErbB4, whereas Nrg-2 binds with >1000-fold greater affinity to ErbB4 (Jones et al., 1999). In addition, Nrg-3 and Nrg-4 bind only to

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ErbB4 (Harari et al., 1999; Jones et al., 1999; Zhang et al., 1997). Because ErbB3, ErbB4, and ErbB2 are each expressed by skeletal muscle fibers in vivo (Altiok et al., 1995; Moscoso et al., 1995; Trinidad et al., 2000; Zhu et al., 1995), the muscle cell membrane at the NMJ is responsive, in principle, to each Nrg. We sought to determine whether Nrg-2, Nrg-3, or Nrg-4 might regulate synapse-specific transcription. Muscle cell lines express ErbB3 and ErbB2 but not ErbB4 (Zhu et al., 1995). Therefore, we first generated a muscle cell line that expresses ErbB4 as well as ErbB3 and ErbB2. We stimulated ErbB3/ErbB2- or ErbB4/ErbB3/ ErbB2-expressing myotubes with each Nrg and found that only Nrg-1 activated AChR transcription in ErbB3/ErbB2-expressing myotubes, whereas Nrg-1 and Nrg-2 each activated AChR transcription in ErbB4/ErbB3/ErbB2-expressing myotubes. Furthermore, we found that Nrg-2 is expressed in motor neurons and terminal Schwann cells and concentrated at synaptic sites, largely adjacent to terminal Schwann cells. Thus, Nrg-2 is a candidate for a signal that regulates synaptic differentiation.

Results Nrg-2 activates AChR transcription in muscle cells expressing ErbB4 as well as ErbB3/ErbB2 Nrg-1 stimulates expression of AChR subunit genes in cultured muscle cells, and this activity can be measured by stimulating

muscle cell lines, stably transfected with AChR-hGH gene fusions, with Nrg-1 and measuring the levels of hGH secreted into the culture medium (Jo et al., 1995; Simon and Burden, 1993). All Nrgs contain an epidermal growth factor (EGF)-like domain, which is necessary and sufficient to bind and activate ErbB receptors. Alternative splicing of nrg-1 and nrg-2 RNAs generates two different EGF-like domains, a or h. Nrg-1h and Nrg-2h have a higher affinity for ErbB receptors than Nrg-1a and Nrg-2a (Jones et al., 1999; Pinkas-Kramarski et al., 1998). We treated Sol8 myotubes, stably transfected with an AChR y subunit-hGH gene fusion, with the EGF domain of Nrg-1h, Nrg-2h, Nrg-3, or Nrg-4, fused to GST, and we measured the levels of hGH secreted into the culture medium with a radioimmunoassay. Control experiments with GST alone showed that it had a small effect on AChR gene expression on this assay (1.1- to 1.2-fold induction over control) (R. Dakour and M.R., data not shown). We found that only GSTNrg-1h induced a dose-dependent increase in AChR gene expression in Sol8 myotubes (Figs. 1A – D). Thus, Nrg-1h is unique among the Nrgs in its ability to activate AChR transcription in Sol8 myotubes. Sol8 myotubes, as well as other muscle cell lines, express ErbB3 and ErbB2, but little, if any, ErbB4 (Jo et al., 1995; Moscoso et al., 1995; Zhu et al., 1995). The postsynaptic membrane at the NMJ, however, contains ErbB4, as well as ErbB3 and ErbB2 (Altiok et al., 1995; Moscoso et al., 1995; Zhu et al., 1995) (but see Trinidad et al., 2000). Thus, to more accurately simulate the response of the postsynaptic membrane to Nrg-1 and to

Fig. 1. ErbB4 is essential for Nrg-2 to stimulate AChR transcription. Sol8 myotubes, stably transfected with an AChR d subunit-hGH gene fusion, and expressing either ErbB2 and ErbB3 (Sol8), or ErbB2, ErbB3, and ErbB4 (Sol8ErbB4), were treated with the EGF-like domain of Nrg-1h, Nrg-2h, Nrg-3 or Nrg-4, and the amount of hGH secreted into the culture medium was measured after 48 h. Only Nrg-1h induces AChR transcription in ErbB3/2-expressing Sol8 myotubes (filled-diamonds). In ErbB4/3/2-expressing Sol8 myotubes, Nrg-1h and Nrg-2h activate AChR transcription to a similar extent; neither Nrg-3 nor Nrg-4 increase AChR gene expression (open squares). The means F standard errors of the means (SEM) are given; the SEMs were calculated from three or more experiments at each concentration of ligand. (*) P < 0.05, two-sided t test.

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determine whether expression of ErbB4 might alter the response of AChR genes to other Nrgs, we force-expressed ErbB4 in Sol8 myotubes (Fig. 2). We treated ErbB4/3/2-expressing Sol 8 myotubes, stably transfected with an AChR y-hGH gene, with GST-Nrg-1h and measured the level of hGH secreted into the culture medium. Fig. 1 shows that GST-Nrg-1h stimulates AChR expression similarly in ErbB3/ 2- and ErbB4/3/2-expressing myotubes, indicating that expression of ErbB4 does not alter the response to Nrg-1h (Fig. 1A). ErbB4 expression, however, alters the response to GST-Nrg-2, as ErbB4/ 3/2-expressing Sol 8 myotubes respond to GST-Nrg-2h (Fig. 1B). Indeed, GST-Nrg-1h and GST-Nrg-2h are similarly effective in inducing AChR transcription in ErbB4/3/-2-expressing myotubes. In contrast, neither GST-Nrg-3 nor GST-Nrg-4 induces AChR expression in ErbB4/3/2-expressing myotubes (Figs. 1C, D). Thus, myotubes that express ErbB4, ErbB3, and ErbB2, mimicking the ErbB expression profile of the postsynaptic membrane, respond similarly to Nrg-1h and Nrg-2h. Fig. 2. ErbB receptor expression in Sol8 and Sol8ErbB4 myotubes. Individual ErbBs were immunoprecipitated from lysates of Sol8 myotubes expressing ErbB3 and ErbB2 (Sol8) or from lysates of Sol8 myotubes transfected with ErbB4 (Sol8ErbB4), and Western blots of the immunoprecipitated proteins were probed with antibodies specific for ErbB2, ErbB3 or ErbB4 (see Methods). Sol8 myotubes express ErbB2 and ErbB3 but not ErbB4, whereas Sol8 myotubes, transfected with ErbB4, express all three ErbBs. A non-specific, smaller band is recognized by the antibody against ErbB4 in lysates from both cell lines. Scans of the film showed a 5.6-fold increase in ErbB3 levels, and a 1.2-fold increase in ErbB2 levels in Sol8ErbB4 myotubes relative to Sol8 myotubes.

Nrg-2 stimulates tyrosine phosphorylation of ErbB receptors We studied ErbB phosphorylation in ErbB3/2- and ErbB4/3/2expressing myotubes to determine whether the ability of the different Nrgs to stimulate AChR transcription correlated with their ability to stimulate ErbB phosphorylation. We treated ErbB3/2and ErbB4/3/2-expressing myotubes with each GST-Nrg, immunoprecipitated individual ErbBs, and probed Western blots with antibodies to phosphotyrosine. In ErbB3/2-expressing myotubes, only GST-Nrg-1h stimulates strong tyrosine phosphorylation of

Fig. 3. Nrg-1 and Nrg-2 stimulate tyrosine phosphorylation of each ErbB in ErbB4/3/2-expressing myotubes. (A) In ErbB3/2-expressing myotubes, only Nrg-1 stimulates tyrosine phosphorylation of both ErbBs (representative results from three independent experiments). (B) In ErbB4/3/2-expressing myotubes, both Nrg-1 and Nrg-2 stimulate tyrosine phosphorylation of each ErbB (representative results from four independent experiments).

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both ErbBs (Fig. 3A). Expression of ErbB4 alters the response to Nrg-2h, as GST-Nrg-2h and GST-Nrg-1h stimulate tyrosine phosphorylation of each ErbB in ErbB4/3/2-expressing myotubes (Fig. 3B). In contrast, myotubes expressing all three ErbBs remain largely unresponsive to Nrg-3 and Nrg-4. Thus, only Nrg-1h and Nrg-2h stimulate phosphorylation of each ErbB. These data indicate that there is correspondence between the ability of each Nrg to stimulate ErbB tyrosine phosphorylation and to induce AChR transcription in ErbB2/3/4-expressing myotubes. Nrg-2 expression in the spinal cord Because Nrg-2, like Nrg-1, activates AChR genes in myotubes expressing ErbB4, as well as ErbB3 and ErbB2, we examined whether Nrg-2 is expressed in motor neurons and concentrated at neuromuscular synapses. We used in situ hybridization to determine whether nrg-2 is expressed in motor neurons. Nrg-1 is expressed widely in the developing embryo but highly expressed in developing and adult motor neurons (Figs. 4A, B, D, E) (Corfas et al., 1995; Meyer et al., 1997), which are situated in the ventral lateral horn of the spinal cord (Fig. 4). We probed sections of the adult rat spinal cord with RNA probes specific for nrg-2 and found that nrg-2 RNA is expressed throughout the cell body-rich, gray matter of the spinal cord, including the ventral lateral region (Fig. 4C). Moreover, this expression pattern is evident, though less robust, as early as E17 (Fig. 4I). Large cells in the ventral horn express both nrg-1 and nrg-2 (Figs. 4D, E, F), indicating that nrg-2 RNA is expressed in motor neurons. To provide a more definitive assessment of Nrg-2 expression in motor neurons, we produced antibodies to a peptide sequence in the

Fig. 5. Antibodies to Nrg-2 do not cross-react with Nrg-1, Nrg-3, or Nrg-4. Purified GST-Nrg fusion proteins were fractionated by SDS-PAGE, and Western blots were probed with affinity-purified antibodies to Nrg-2. GSTNrg-2h, but neither GST-Nrg-1h, GST-Nrg-3, nor GST-Nrg-4 are recognized by the antibodies to Nrg-2. Identical results were obtained following longer exposures (data not shown). The stripped blot was re-probed with antibodies to GST to confirm equivalent loading of each GST-Nrg. The position and size (kDa) of protein standards are indicated to the left.

EGF-like domain of Nrg-2, which is present in Nrg-2a and Nrg-2h isoforms, but absent from other Nrgs. We assessed the specificity of the antibodies to Nrg-2 by probing Western blots of recombinant Nrg-1h, Nrg-2h, Nrg-3, and Nrg-4. Fig. 5 shows that these antibodies recognize Nrg-2h, but neither Nrg1h, Nrg-3, nor Nrg-4. On whole-brain extracts from adult rat, a tissue known to express Nrg-2 mRNA at high levels (e.g., Carraway et al., 1997), the antibody recognized two bands that were absent when it was pre-incubated with the immunizing peptide before blotting (panel B, Supplementary material). At least two Nrg-2 transcripts were detected by RT-

Fig. 4. nrg-2 mRNA is expressed by motor neurons. (A, B, C) Low-magnification views of three adult rat spinal cord cross-sections (cervical region) hybridized for crd-nrg-1, ig-nrg-1, and nrg-2, respectively. Nrg-2 is expressed throughout the gray matter, including in the cell bodies of neurons in the ventral horn, which express crd-nrg-1 and ig-nrg-1. (D, E, F) High magnification views of the ventral horn of three adult rat spinal cord cross-sections hybridized for crd-nrg-1, ig-nrg-1, and nrg-2, respectively. The cell bodies of large cells in the ventral horn, presumably motor neurons, express nrg-1 and nrg-2 mRNA. (I) Although less robust, the adult pattern of nrg-2 expression is also evident in embryos (E17). Scale bar: (A, B, C, G, H, I) 360 Am; (D, E, F), 100 Am.

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PCR from brain (panel A, Supplementary material), which may account for the bands observed in the Western blot. Multiple Nrg-2 RNA isoforms in adult rat brain have been detected previously by others (Yamada et al., 2000), and three distinct full-length transcripts of approximately 7, 4.5, and 3.4 kb, respectively, were observed in adult murine brain (Carraway et al., 1997). Although, the precise size of the Nrg-2 proteins in the brain is unknown, the amino acid sequences deduced from the different cDNAs predict molecular weights that range from approximately 94 kDa for the transmembrane precursors to approximately 45 kDa for the smallest fully processed forms. This range is within the sizes observed in our Westerns (panel B, Supplementary material). We stained sections of adult rat spinal cord with affinitypurified antibodies to Nrg-2 and with antibodies to choline acetyl transferase (ChAT), which marks motor neurons. Fig. 6 shows that large, ChAT-positive neurons in the ventral, lateral horn of the

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spinal cord are stained by antibodies to Nrg-2 (Figs. 6A, B, C). At higher magnification, it is apparent that Nrg-2 is present in processes, as well as in cell bodies of these ChAT-positive neurons (Figs. 6D, E, F). ChAT and Nrg-2, however, appear to be concentrated in distinct subcellular compartments, as ChAT and Nrg-2 staining show imperfect overlap (Figs. 6G, H, I). Nearly all (144/146, 98.6%) of the ChAT-positive cells stained for Nrg-2. In addition to motor neurons, cells that are in more medial and dorsal regions of the spinal cord and that lack ChAT expression, are stained with antibodies to Nrg-2 (Figs. 6A, B, C). Pre-incubation of the antibody with the immunizing peptide drastically reduced the staining in motor neurons (Figs. 6J, K, L) and in other neurons (data not shown). Thus, the pattern of Nrg-2 protein expression is consistent with the pattern of nrg-2 RNA expression (Fig. 4), indicating that Nrg-2 protein is present in motor neurons, as well as in other spinal cord neurons.

Fig. 6. Nrg-2 protein is expressed in motor neurons in the adult spinal cord. Cross-sections of adult rat spinal cord were stained with antibodies to ChAT, to mark motor neurons, and with affinity-purified antibodies to Nrg-2. (A, B, C) Large neurons in the ventral, lateral horn of the spinal cord, which stain for ChAT, also stain for Nrg-2 (arrows); in addition, neurons in more dorsal and medial regions of the spinal cord stain for Nrg-2 but not for ChAT. (D, E, F) At higher magnification, Nrg-2 staining is evident in the cell bodies and processes of ChAT-positive neurons. (G, H, I) The subcellular distribution of ChAT and Nrg-2 overlap imperfectly. (J, K, L) Pre-incubation of Nrg-2 antibody with immunizing peptide drastically reduces staining of motor neurons. Scale bar: (A, B, C), 120 Am; (D, E, F, K, L), 64 Am; (G, H, I), 30 Am.

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Nrg-2 at the NMJ We stained sections of adult rat skeletal muscle with affinitypurified antibodies to Nrg-2 to determine whether Nrg-2 is concentrated at NMJs. Synaptic sites were marked by co-staining with antibodies to the synaptic vesicle protein SV2 and with fluorescein-a-bungarotoxin to label AChRs. We found that Nrg-2 is highly concentrated at synaptic sites (Figs. 7A, B, C). Nrg-2 staining, however, does not overlap precisely with SV2 or AChR staining, indicating that Nrg-2 is concentrated neither in presynaptic terminals nor in the postsynaptic membrane (Figs. 7D, E, F, G). Instead, Nrg-2 staining accumulates adjacent to nerve terminals, possibly associated with terminal Schwann cells and/ or in the extracellular matrix adjacent to the synaptic cleft (see below). To determine whether expression of synaptic Nrg-2 is innervation-dependent, we denervated skeletal muscle and stained sections

of muscle with antibodies to Nrg-2. Fig. 7 demonstrates that synaptic expression of Nrg-2 is indeed innervation-dependent, as Nrg-2 staining is lost following denervation (Figs. 7H, I). Preincubation of the antibody with the immunizing peptide drastically reduced the staining at NMJs (Figs. 6J, K, L). To investigate further whether Nrg-2 is associated with terminal Schwann cells, we simultaneously stained sections of skeletal muscle with antibodies to Nrg-2, antibodies to S-100, a marker for Schwann cells, and a-bungarotoxin (Figs. 8A – D). These triplestaining experiments confirm that Nrg-2 is concentrated adjacent to terminal Schwann cells (Figs. 8 A – D), suggesting that terminal Schwann cells synthesize Nrg-2. To determine whether Nrg-2 is indeed expressed by terminal Schwann cells, we hybridized longitudinal and cross-sections of skeletal muscle with RNA probes specific for nrg-2. Fig. 8F shows that nrg-2 mRNA is expressed in cells, like terminal Schwann cells, positioned in the endplate zone within the central region of the muscle. We hybrid-

Fig. 7. Nrg-2 protein accumulates at NMJs. (A, B) Cross-sections of adult rat sternomastoid muscle were stained with a monoclonal antibody to SV2 to label nerve terminals (A) and with affinity-purified antibodies to Nrg-2 (B). (C) Nrg-2 staining is concentrated at all synaptic sites, but does not overlap precisely with SV2 staining. (D, E, F) A higher magnification view of a NMJ (outlined in C) shows that Nrg-2 staining extends beyond the boundaries of SV2 staining and appears to outline a cell body of a terminal Schwann cell. (G) A cross-section of adult rat sternomastoid muscle was labeled with fluorescein-abungarotoxin to mark AChRs (green) and with affinity-purified antibodies to Nrg-2 (red); Nrg-2 staining is adjacent to AChRs. (H, I) Cross-sections of a denervated (3-day) adult rat soleus muscle were stained for Nrg-2 (H) and AChRs (I); synaptic Nrg-2 staining is lost after denervation, demonstrating that synaptic expression of Nrg-2 is innervation-dependent. (J, K, L) Pre-incubation of Nrg-2 antibody with immunizing peptide (pep) drastically reduces staining near normal NMJs. Scale bar: (A, B, C), 55 Am; (D, E, F, G, J, K, L), 20 Am; (H, I), 32 Am.

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2 accumulates near synaptic sites, where it has the potential to activate ErbBs in the muscle or terminal Schwann cells. Nrg-2 expression in the D6P2T Schwann cell line To provide additional support for our findings that terminal Schwann cells synthesize Nrg-2, and to further characterize our antibodies, we examined Nrg-2 mRNA and protein expression in the model Schwann cell line D6P2T (Bansal and Pfeiffer, 1987). We detected a strong signal for Nrg-2 mRNA in D6P2T cells by RT-PCR (panel A, Supplementary material). While two bands were detected in brain RNA, only a single band was observed in the Schwann cell line RNA (panel A, Supplementary material). DNA sequencing confirmed the identity of the bands as Nrg-2 cDNAs (data not shown). A single, specific protein band that migrated similarly to one of the bands in the brain extract was found as recognized by our Nrg-2 antibodies in D6P2T extracts (panel B, Supplementary material). Thus, these results show that cultured Schwann cells lines can synthesize Nrg-2 mRNA and protein and are consistent with our findings that terminal Schwann cells do so in vivo.

Discussion

Fig. 8. Nrg-2 is expressed by terminal Schwann cells at the adult NMJ. Cross-sections of adult rat tibialis anterior muscle were stained with antibody to Nrg-2 (A), a monoclonal antibody to S100 (C), and Alexa-647a-bungarotoxin, to mark synaptic sites (B). Nrg-2 staining accumulates on the surface of a terminal Schwann cell body that faces away from the synaptic site, as well as on terminal Schwann cell processes (D). Longitudinal sections of adult rat tibialis anterior muscle were processed for in situ hybridization with digoxigenin-labeled antisense riboprobes for AChR a subunit (E) and nrg-2 (F). En-face view shows signal for AChRa around clustered synaptic myonuclei of a single NMJ (E), whereas signal for nrg-2 accumulates around one or two non-muscle cells (arrowhead). These images were taken from the synaptic region of two separate muscles. Cross-sections of adult rat soleus muscle were processed for nrg-2 in situ hybridization as above (H), and serial sections were stained for cholinesterase (ChE) to mark synaptic sites (arrow in G). nrg-2 RNA (arrowhead in H) is expressed in a non-muscle cell, positioned like terminal Schwann cells, on the presynaptic site of the ChE-stained synaptic cleft (arrow in G and H). Sense probes yielded no signal (data not shown). Scale bar: (A – D), 5 Am; (E, F), 20 Am; (G, H), 12 Am.

ized cross-sections of skeletal muscle with a nrg-2 probe and stained serial cross-sections for AChE activity, which marks the synaptic cleft and thereby provides a convenient border for assigning the source of nrg-2 expression. Fig. 8H shows that nrg-2 RNA is contained in cells that reside presynaptic to the AChE stain, indicating that Schwann cells rather than myofibers express nrg-2. Thus, Nrg-2 protein and RNA expression patterns are consistent with the idea that terminal Schwann cells synthesize Nrg-2. As most of the Nrg-2 staining near synaptic sites is associated with terminal Schwann cells, and because staining is absent following denervation (Fig. 7H), these data indicate that expression of Nrg-2 by terminal Schwann cells is dependent upon innervation. Taken together, these results support the idea that Nrg-

Here, we show that Nrg-2 is expressed by adult motor neurons and terminal Schwann cells and is concentrated near synaptic sites. Moreover, we show that the EGF domain of Nrg-2h, fused to GST, stimulates ErbB phosphorylation and activates AChR transcription in cultured myotubes expressing ErbB4, ErbB3, and ErbB2. Because ErbB4, together with ErbB3 and ErbB2, is expressed by muscle fibers in vivo and is concentrated in the synaptic muscle membrane, Nrg-2 is a candidate for a signal that regulates synaptic differentiation. We find that Nrg-2 activates AChR transcription in myotubes expressing ErbB4, ErbB3, and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Although ErbB3 expression is elevated 5.6-fold in ErbB4/3/2-expressing myotubes (Fig. 2), this increase in ErbB3 expression is unlikely to account for their response to Nrg-2 (Fig. 1). Importantly, biochemical studies have shown that Nrg-2 binds poorly to ErbB3 and with 1000-fold greater affinity to ErbB4/2 than ErbB3/2 heterodimers (Jones et al., 1999). Consistent with these data, we find that the same induction in AChR expression (1.5-fold) is elicited in ErbB3/2expressing myotubes by 1000 ng/ml GST-Nrg-2 and in ErbB4/3/2expressing myotubes by 5 ng/ml GST-Nrg-2. It is unlikely that this 200-fold difference in the amount of Nrg-2 needed to induce the same response in both cell populations is explained by the 5.6-fold increase in ErbB3 expression observed in the ErbB4/3/2-expressing myotubes. Rather, it would appear better explained by the larger affinity of Nrg-2 for ErbB4 than for ErbB3. Taken together, these data indicate that the sensitivity of ErbB4/3/2-expressing myotubes to Nrg-2 is dependent upon ErbB4 expression. Because muscle cell lines express little if any ErbB4 (Zhu et al., 1995), this dependence on ErbB4 expression may account for the failure of prior studies to detect a role for Nrg-2 in stimulating AChR transcription (Busfield et al., 1997). Consistent with these data, we find that Nrg-1, but neither Nrg-2, Nrg-3, nor Nrg-4 stimulates ErbB tyrosine phosphorylation in myotubes expressing ErbB3 and ErbB2. In myotubes expressing ErbB4, ErbB3 and ErbB2, Nrg-2 and Nrg-1 each stimulates tyrosine phosphorylation of all three

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ErbBs. Previous studies demonstrated that ErbB4 binds Nrg-2 with a greater affinity than either Nrg-3 or Nrg-4 (Harari et al., 1999; Jones et al., 1999; Zhang et al., 1997), and consistent with these data, we find that Nrg-3 and Nrg-4 induce only marginal ErbB phosphorylation in myotubes expressing all three ErbBs. Although these results suggest that differences in the affinity of Nrgs and ErbBs may account for the failure of Nrg-3 and Nrg-4 to stimulate AChR transcription, it is also possible that different Nrgs stimulate phosphorylation of distinct tyrosine residues on ErbB4 (Sweeney et al., 2000), leading to differing biological responses. Thus, although Nrg-4 stimulates tyrosine phosphorylation of ErbB4, the phosphorylated tyrosine(s) may not serve as a docking site for a signaling complex leading to an increase in AChR transcription. In addition, as each of the 4 Nrgs can stimulate robust ErbB4 phosphorylation in primary cerebellar granule neurons (A.L.P, unpublished observations), receptor activation may be dependent upon the presence of other molecules that influence ErbB signaling (Carraway and Sweeney, 2001) or to differences in the subcellular distribution of the receptors (Zhao et al., 1999). Nrg-2 mRNA and protein are expressed by motor neurons, additional cell types in the adult spinal cord, and terminal Schwann cells at the NMJ. Nrg-2 protein accumulates adjacent to synaptic sites in skeletal muscle, and this accumulation is dependent upon innervation. We also found that the Schwann cell line D6P2T expresses Nrg-2 mRNA and protein. Schwann cell-derived Nrg-2 appears suitably positioned to act on the perisynaptic domain of the NMJ, where it could activate ErbB receptors on the muscle fibers or on the Schwann cells themselves. To reach ErbB receptors in the muscle surface, Schwann cell-derived Nrg-2 would have to diffuse from its site of accumulation on the surface of the terminal Schwann cell that faces away from the synapse. Ig-Nrg-1 that apparently accumulates within the synaptic basal lamina has also been proposed to diffuse to the synaptic sarcolemma to activate ErbB receptors there (Trinidad et al., 2000). It is unclear whether perisynaptic signals contribute to synapse-specific transcription, but the density of AChRs and AChR transcription are substantially greater in the perisynaptic than in the extrasynaptic region of the muscle (Salpeter et al., 1988; Simon et al., 1992). The pattern of synapse-specific transcription is broadened in E18.5 mice that lack Schwann cells (Woldeyesus et al., 1999), indicating that Schwann cells are not essential, but may contribute to synapse-specific transcription during embryogenesis. As these mutant mice die at birth, a potential role for terminal Schwann cells in regulating synapse-specific transcription at mature synapses in adult mice could not be examined (Woldeyesus et al., 1999). Although Nrg-2 accumulates adjacent to synaptic sites, we cannot exclude the possibility that motor neuron-derived Nrg-2 is present within the synaptic cleft, at levels that are too low to detect with existing antibodies. Thus, it is possible that neuronal Nrg-2 activates ErbB receptors in the postsynaptic muscle membrane. Because Nrg-2 staining accumulates on the terminal Schwann cell surface and disappears following denervation, it is tempting to speculate that Nrg-2 signaling might normally contribute to prevent the ‘‘activation’’ of Schwann cells, which occurs following denervation. ‘‘Activated’’ terminal Schwann cells display morphological changes likely resulting from poorly characterized changes in gene expression. Absent Nrg-2’s ‘‘restrain’’, terminal Schwann cells might acquire the activated phenotype without having to denervate the muscle. Neuronal Nrg-1 fulfills several criteria expected for a signal that activates synapse-specific transcription. Selective inactivation of

motor neuron- and sensory neuron-derived Nrg-1 demonstrates, however, that neuronal Nrg-1 is dispensable for synapse-specific AChR transcription during embryogenesis (Yang et al., 2001). As synapse-specific expression during development is dependent upon Agrin/MuSK signaling (DeChiara et al., 1996; Gautam et al., 1996), Agrin may be the neural signal that induces synapsespecific transcription during embryogenesis. Additional signals may regulate synapse-specific transcription at mature synapses in postnatal mice. Our findings raise the possibility that Nrg-2, which is expressed by motor neurons and terminal Schwann cells and activates AChR transcription in cultured myotubes, has a role in stimulating gene expression or regulating additional aspects of synaptic maturation.

Experimental methods Isolation of ErbB4-expressing muscle cell lines Sol 8 myoblasts were co-transfected with an expression vector encoding human ErbB4, under the control of regulatory elements from the myosin light chain gene, and a CMV-neomyocin expression vector. We isolated clones of stably transfected myoblasts and screened myotubes derived from the myoblast lines for ErbB4 expression by Western blotting. Myoblasts, stably transfected with ErbB4, were subsequently co-transfected with an AChR y subunit ( 1823/+25)-hGH gene fusion and a CMV-hygromycin expression vector, and pooled stably transfected myoblasts were selected in hygromycin. Myoblasts were selected and induced to differentiate into myotubes as described previously (Jo et al., 1995; Simon and Burden, 1993). Stably transfected cells were treated with recombinant Nrgs for 48 h, and the amount of hGH secreted from treated and untreated cells was measured by a radioimmunoassay as described previously (Jo et al., 1995). Production of recombinant neuregulin factors Recombinant neuregulins (Nrgs) were produced in High Five cells as glutathione S-transferase (GST) fusions with the EGF-like motif of either Nrg-1 (h1 form), Nrg-2 (h1-like form), Nrg-3, or Nrg-4. Briefly, the approximately 35 kDa recombinant proteins (see Fig. 5) were produced by infecting approximately 2  107 High Five cells with approximately 5  107 – 2  108 pfu recombinant virus at 27jC for 4 days (Carraway et al., 1997). The culture media was harvested, cellular debris was removed by centrifugation, and the supernatant was incubated with glutathioneSepharose (Amersham Pharmacia, Piscataway, NJ). The beads were washed with PBS, and the fusion proteins were eluted with 10-mM glutathione. Eluted proteins were stabilized with BSA (100 mg/ml), dialyzed into PBS, and concentrated. The concentration of GST-Nrgs was estimated by comparative Coomassie staining after SDS-PAGE. The Nrg-1h EGF-like domain cassette has the sequence: TSHLIKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQK* The Nrg-2h EGF-like domain cassette has the sequence: SGHARKCNETAKSYCVNGGVCYYIEGINQLSCKCPVGYTGDRCQQFAMVNFSKHLGFELKEAEELYQK*

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The Nrg-3 EGF-like domain cassette has the sequence:

Reverse transcriptase-polymerase chain reaction (RT-PCR)

SEHFKPCRDKDLAYCLNDGECFVIETLTGSHKHCRCKEGYQGVRCDQFLPKTDSILSDPTDHLGIEFMESEDVYQR*

Adult rat brain RNA was purchased from Clontech. D6P2T cells were grown to confluence in DMEM supplemented with 10% FBS and 50 Ag/ml gentamycin. Total RNA was prepared with RNA STAT-60 reagent according to the manufacturer’s instructions (TelTest, Friendswood, TX). SuperScript II reverse transcriptase (Invitrogen, CA) was used to make cDNA from 2 Ag of RNA per sample, and 1/10th of the reaction was used for PCR with HotStart PCR Master Mix (Qiagen, San Diego, CA). The following were the Nrg-2 primers used: Forward: 5V-AGAACTCACG(A/G)CTACAGTTCAAC-3V. Reverse: 5V-TCTTGCAGTAGGC(C/G)ACCACACAGAC-3V. The degenerate bases in the middle of each primer allow for amplification of rat- or mouse-derived templates. The cycling parameters were: 40 cycles with 1 min 95jC denaturation, 1 min 56jC annealing, 30 s 72jC extension. Samples were run in 2% agarose gels. Amplified bands were cloned and their identity was confirmed by DNA sequencing.

The Nrg-4 EGF-like domain cassette has the sequence: TDHEQPCGPRHRSFCLNGGICYVIPTIPSPFCRCIENYTGARCEEVFLPSSSIPSE* Animals and denervation Surgical procedures and animal care were approved by institutional authorities. Adult rats of the Wistar, AO, and Sprague – Dawley strains were used. Immunohistochemistry was performed on the sternomastoid, tibialis anterior, and soleus muscles. Leg muscles were denervated by cutting the sciatic nerve under anesthesia. One hundred Al of a 75 mg/ml ketamine, 5 mg/ml xylazine mixture were administered i.p. for every 100 g in weight. Animals were sacrificed 3 – 4 days after denervation, and muscles were removed and processed as described below. In situ hybridization Radioactive in situ hybridization was performed essentially as previously described (Simmons et al., 1989), with minor modifications. Adult rats were perfused for 20 min with 4% paraformaldehyde in 0.1 M sodium borate buffer, pH 9.5. The spinal cord was post-fixed for 3 days and cryoprotected in 15% sucrose, 0.1 M phosphate buffer, pH 7.2, for 24 h before storage at 70jC. Coronal sections (25 Am) were cut on a cryostat and mounted on gelatin and poly-L-lysine-coated slides. The pre- and post-hybridization procedures were performed as described previously (Simmons et al., 1989), except the sections were additionally post-fixed (10% buffered formalin) for 30 min, followed by four 5-min washes in 0.05 M KPBS before the pre-hybridization steps. We used PCR to amplify Nrgs: (1) a 767 bp fragment (nucleotides #555-1321, as reported in accession AF194438) from the CRD isoform of Nrg-1, (2) a 501 bp fragment (nucleotides #45-545, as reported in accession U02324) from the typeI/II Igisoform of Nrg-1: (3) a 751 bp fragment (nucleotides #4321182, as reported in accession D89995) from Nrg-2. These fragments were subcloned into the EcoRI/BamH1 site of pBluescript (SK-). After linearization of these constructs with EcoRI, T3 polymerase (Promega) was used to generate antisense transcripts. Transcriptions were performed using 125 ACi 33P-UTP (2000 – 4000 Ci/mmol, NEN, Boston, MA). After hybridization, the sections were defatted in xylene, rinsed first in 100% ethanol, then 95% ethanol, air dried and dipped in NTB2 emulsion (Kodak, Rochester, NY) diluted 1:1 with water. The slides were exposed for 2 – 5 weeks and developed in Kodak D19 developer. In situ hybridization with digoxigenin-labeled probes was performed essentially as previously described (Braissant and Wahli, 1998); chemically hydrolyzed riboprobes of about 200 bp were used (Schaeren-Wiemers and Gerfin-Moser, 1993). The probe used for the AChRa subunit was described previously (e.g., Yang et al., 2001). Cholinesterase activity was visualized by histochemical staining (Karnovsky, 1964).

Antibodies and immunohistochemistry Antibodies to Nrg-2 were produced by immunizing rabbits with an 11-amino acid sequence (NH2 – CYYIEGINQLS – COOH) in the EGF domain of Nrg-2 coupled to KLH as carrier, and the antiserum was affinity-purified on a peptide affinity column using the SulfoLink kit (Pierce, Rockford IL) according to the manufacturer’s instructions. These antibodies were used at 1/20 – 1/40 for immunocytochemistry and at 1/1000 for Western blots. Polyclonal antibodies to choline acetyltransferase (ChAT) (AB144P; Chemicon International, Temecula, CA) were used at 1/200. A monoclonal antibody to SV2 (Developmental Studies Hybridoma Bank, University of Iowa) was used at 1/200, and a monoclonal antibody to S100 (Clone SH-B1, Sigma, St. Louis, MO) was used at 1/300. Rabbit antiserum to a GST-Nrg-2h fusion protein, which recognizes GST and not Nrg-2h (data not shown), was used at 1/ 30 000. Fluorescein- and peroxidase-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA). Nrg-2 staining was detected with biotin-conjugated secondary antibodies (Jackson Immunoresearch and Molecular Probes, Eugene, OR) and Cy5-strepavidin (Jackson Immunoresearch) or Alexa-568 strepavidin (Molecular Probes). Fluorescein- and Alexa-647-a-bungatoxin, used to mark AChRs, were purchased from Molecular Probes. Frozen sections of unfixed spinal cord and skeletal muscle were stained as described previously (Rimer et al., 1998). Biotinconjugated secondary antibodies and fluorochrome-conjugated strepavidin were added together during the secondary incubation. For antigen blocking experiments, antibody against Nrg-2 was incubated with immunizing peptide (10 Ag/ml in PBS) overnight at 4jC or for 2 h at room temperature before the staining procedure. ErbB phosphorylation Sol8 myotubes were serum-starved, stimulated with Nrgs (250 ng/ml) for 5 – 10 min, and lysed in a buffer (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, and a mixture of protease and phosphatase inhibitors. For immunoprecipitation, equal amounts of protein from the cell lysates were incubated for 2 h or overnight at 4jC with either antibodies to ErbB2 (Ab-3, Oncogene, San Diego, CA, and rabbit anti-mouse #61-6500, Zymed, South San Francisco, CA), antibodies to ErbB3 (C-17, Santa Cruz Biotech-

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nology, Santa Cruz, CA, or affinity-purified rabbit antibodies #4121, C. Lai), or antibodies to ErbB4 (#616, (Zhu et al., 1995)). Immune complexes were collected using Protein G-agarose or Protein A-agarose and washed three times with cold lysis buffer. Immunoprecipitated proteins were eluted from the agarose beads, resolved by SDS-PAGE, and transferred to PVDF membranes. Western blots were blocked with buffered BSA, probed with antibodies to phosphotyrosine (4G10, Upstate, Lake Placid, NY, or RC-20, BD Transduction Laboratories, San Diego, CA), probed with peroxidase-conjugated secondary antibodies; antibody-binding was detected by chemiluminescence. Membranes were stripped, blocked with buffered nonfat dried milk, and re-probed with the above antibodies to ErbB receptors; in some experiments, rabbit polyclonal antibodies to ErbB2 (sc-284, Santa Cruz Biotechnology) were used to probe Western blots. Protein extracts and Western blots of brain tissue and D6P2T cells Whole brain from 2-month old rat was homogenized with a polytron in a ice-cold buffer containing 50 mM Tris pH 7.4, 2 mM EDTA, 10 Ag/ml aprotinin and leupeptin, 25 Ag/ml pepstatin A. A ratio of 25 ml of buffer/g of tissue was used. The extract was passed through a 21-gauge needle several times to reduce viscosity and stored at 80jC until use. D6P2T cells were cultured as above, rinsed in cold PBS, and scraped from the dish in 50 mM Tris pH 7.4, 2 mM EDTA, 10% v/v Protease Inhibitor cocktail (Sigma); cells were broken up by passage of the suspension through a 23gauge needle several times. Extract was spun at 14,000  g for 15 min and the supernatant was concentrated using a Centricon YM10, 10,000 MW cut-off (Millipore Corp., Bedford, MA), following the manufacturers’ instructions. Protein concentration was estimated using Bradford-based, Biorad Protein Assay (Biorad, Hercules, CA). The indicated amount of extracts were mixed with 2 Laemmli sample buffer (Sigma), heated for 5 min at 95jC, and fractionated in 7.5% acrylamide denaturing gels. Transfer to PVDF membranes and Western blotting was carried out as above. Affinity-purified Nrg-2 antibody was used at 1/1000 – 1/2000 dilution. Horse Radish Peroxidase-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch) was used at 1/3000 dilution.

Acknowledgments We thank Wes Thompson for the D6P2T cells and for comments on the manuscript. Supported by University of Texas at Austin start-up funds (M.R.), and NIH Grants (NS27963, S.J.B; NS32367, C.L.; GM65797, M.R.).

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