Agrin Promotes Synaptic Differentiation by Counteracting an Inhibitory Effect of Neurotransmitter
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Agrin promotes synaptic differentiation by counteracting an inhibitory effect of neurotransmitter Thomas Misgeld*, Terrance T. Kummer*, Jeff W. Lichtman, and Joshua R. Sanes† Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; and Department of Anatomy and Neurobiology, Washington University Medical School, St Louis, MO 63110 Contributed by Joshua R. Sanes, June 9, 2005 Synaptic organizing molecules and neurotransmission regulate bryos than in controls (10), suggesting that ACh could actually synapse development. Here, we use the skeletal neuromuscular antagonize postsynaptic differentiation. junction to assess the interdependence of effects evoked by an Here, using agrnϪ/Ϫ and chatϪ/Ϫ mice that we previously essential synaptic organizing protein, agrin, and the neuromuscu- generated and characterized, we reassess agrin’s role and ask lar transmitter, acetylcholine (ACh). Mice lacking agrin fail to whether ACh is the nerve-derived factor that antagonizes syn- maintain neuromuscular junctions, whereas neuromuscular syn- aptic differentiation. Using double-mutant mice, we show that apses differentiate extensively in the absence of ACh. We now synapses do form in the absence of agrin provided that ACh is demonstrate that agrin’s action in vivo depends critically on cho- also absent. We then provide evidence from cultured muscle linergic neurotransmission. Using double-mutant mice, we show cells that ACh destabilizes nascent postsynaptic sites, and that that synapses do form in the absence of agrin provided that ACh one critical physiological role of agrin is to counteract this is also absent. We provide evidence that ACh destabilizes nascent ‘‘antisynaptogenic’’ influence. postsynaptic sites, and that one major physiological role of agrin Methods is to counteract this ‘‘antisynaptogenic’’ influence. Similar interac- Ϫ/Ϫ Ϫ/Ϫ tions between neurotransmitters and synaptic organizing mole- Mutant Mice. chat and agrn mice were generated in our cules may operate at synapses in the central nervous system. laboratory and have been described in detail (4, 10). acetylcholine ͉ activity ͉ neuromuscular junction ͉ synaptogenesis Histology. Embryos were decapitated and fixed by immersion in 4% paraformaldehyde in PBS. Tail samples were kept for genotyping. Tissues were postfixed for 12–24 h before staining. wo classes of intercellular signals important for synaptogenesis Images were acquired on a Bio-Rad MRC1024 or a FV500 Tare proteins that organize pre- and postsynaptic differentiation Olympus (Melville, NY) confocal microscope and are repre- and neurotransmitters that elicit electrical responses. These signals sented as maximum intensity projections that were contrast- presumably interact to shape the synapse, but they are generally adjusted in PHOTOSHOP (Adobe Systems, San Jose, CA). Num- studied separately. At the skeletal neuromuscular junction (NMJ), bers of AChR clusters and muscle fibers were determined from the neurotransmitter is acetylcholine (ACh), and a main synaptic images taken with a ϫ20 objective; AChR cluster size was organizing molecule is the glycoprotein agrin. The aim of the measured from images taken with a ϫ60 objective. present study was to assess the interdependence between ACh and Muscles were stained as described (10) by using the following agrin during synaptic differentiation. reagents: anti-neurofilament (Chemicon), anti-synaptophysin Agrin is synthesized and released by motoneurons at the NMJ, (Zymed), Alexa594-labeled bungarotoxin (Btx) (2.5 g͞ml; In- where it is believed to promote aggregation of ACh receptors vitrogen), Alexa660-phalloidin (Invitrogen), anti-laminin-2 (AChRs) and associated proteins to form a postsynaptic appa- (1117; gift of R. Timpl; Max Planck Institute of Biochemistry, ratus beneath the nerve terminal (1, 2). Agrin-deficient Martinsried, Germany), anti-rapsyn (made in our laboratory), (agrnϪ/Ϫ) mice are born paralyzed because of profound synaptic anti-muscle-specific kinase (MuSK) (gift of M. Ruegg; Biozen- defects (3, 4): myotubes bear dramatically fewer and smaller trum, University of Basel), anti-acetylcholinesterase (gift of T. AChR clusters than in controls, and most of the remaining Rosenberry; Mayo Clinic College of Medicine, Jacksonville, clusters are unapposed by axons (3, 4). These findings supported FL), and anti-SV2 (Developmental Studies Hybridoma Bank, the ‘‘agrin hypothesis,’’ which states that agrin is the master Iowa City, IA). Secondary antibodies were from Jackson Im- nerve-derived initiator of postsynaptic differentiation (1). Sub- munoResearch and Cappel (MP Biomedical, Irvine, CA). Mus- sequent studies, however, showed that immature postsynaptic cle fiber number (Fig. 1d) was determined from rotation of sites form in mice lacking agrin but then abort differentiation confocal stacks through the entire thickness of the muscle. The and disassemble (5, 6), reopening the question of agrin’s func- number of AChR clusters was divided by the number of muscle tion. Moreover, AChR clusters persist longer in muscles lacking fibers to obtain the number of AChR clusters per fiber. MuSK- motor innervation entirely than in muscles innervated by agrnϪ/Ϫ and rapsyn-to-AChR density ratios were measured by determin- axons (5, 6), suggesting that the nerve provides a second factor ing the intensity of fluorescence over background in immuno- that acts to disperse postsynaptic sites. stained cryosections for either antigen and dividing by AChR ACh is a reasonable candidate for the dispersal factor because density. it, or the postsynaptic depolarization it elicits, regulates AChR Tissue Culture. C2C12 myoblasts (American Type Culture Col- subunit gene expression (2, 7) and stimulates AChR endocytosis lection) were cultured as described (9). Reagents were from (8). The existence of complex ‘‘postsynaptic’’ structures on myotubes cultured aneurally demonstrates that ACh is not required for postsynaptic differentiation (9). Moreover, NMJs Abbreviations: ACh, acetylcholine; AChR, ACh receptor; NMJ, neuromuscular junction; CCh, differentiate extensively in mice that lack ACh due to targeted carbamylcholine; Btx, bungarotoxin; En, embryonic day n; MuSK, muscle-specific kinase; mutation of the gene that encodes its sole synthetic enzyme, dko, double knockout. choline acetyltransferase (chatϪ/Ϫ mice; refs. 10 and 11). Indeed, *T.M. and T.T.K. contributed equally to this work. NMJs appear to develop precociously in the absence of neuro- †To whom correspondence should be addressed. E-mail: [email protected]. Ϫ Ϫ transmitter; they are larger and more complex in chat / em- © 2005 by The National Academy of Sciences of the USA 11088–11093 ͉ PNAS ͉ August 2, 2005 ͉ vol. 102 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504806102 Downloaded by guest on September 26, 2021 Fig. 1. Neuromuscular synapses in mice lacking agrin and ACh. (a) E17 diaphragm stained with Btx to label AChRs. Postsynaptic AChR aggregates are missing in agrnϪ/Ϫ muscles but are present in a widened end-plate band both in chatϪ/Ϫ muscles, which lack ACh, and in agrnϪ/Ϫ, chatϪ/Ϫ double mutants (dko). (b) Schematic of results in a and c.(c) E17 triangularis sterni muscles stained with Btx (red) and antibodies to axons (green). Nerves invade the muscle in all four genotypes, but synapses are missing in agrnϪ/Ϫ muscles, whereas they are present in control (ctrl), chatϪ/Ϫ, and dko muscles. Two uninnervated dko AChR clusters are indicated by arrowheads. (d) Quantification of synapse morphology in triangularis sterni. AChR clusters in dko muscles are more similar to those in ctrl and chatϪ/Ϫ muscles than to AChR clusters in agrnϪ/Ϫ muscles in number per muscle fiber, percentage of clusters apposed by an axon (mean ϩ SEM, n ϭ 6–9ϫ20 fields), and size (mean ϩ SEM, n ϭ 4 ϫ60 fields with Ͼ15 AChR clusters each). *, differs from other three genotypes (P Ͻ 0.005, t test). (e) High-power view of nerve–muscle contacts in E17 triangularis sterni muscles. (Scale bars, 50 mina and c;10mine.) Sigma and were used as follows except where indicated: car- muscles examined, including intercostals, diaphragm, and trianu- bamylcholine (CCh; 100 M for 12 h); ␣-Btx (2 g͞ml to block laris sterni (Fig. 1 a–c and Fig. 7 a and b, which is published as all AChRs, or 0.1 g͞ml for 30 min to block Ϸ50% of AChRs supporting information on the PNAS web site). Most dko muscle before CCh treatment); veratridine (250–500 M); scorpion fibers bore a single cluster, which was similar in size to those in toxin from Androctonus australis (100 g͞ml, ICN); QX-314 (500 controls (Fig. 1d and Fig. 7c). Rescue was imperfect in that some M), tetrodotoxin (5 M); curare (1 mM), high potassium (50 dko fibers lacked an AChR cluster, whereas others had multiple mM, substituted for sodium to preserve osmolarity), and cyclo- clusters (Fig. 2g and data not shown). Moreover, the central ͞ Ϫ/Ϫ heximide (20 g ml for 4 h before the addition of CCh and for endplate band was wider in dko and chat muscles than in NEUROSCIENCE another6hinthepresence of CCh; ref. 12). Chinese hamster controls. Nonetheless, the difference between agrnϪ/Ϫ and dko ovary cells expressing membrane-tethered Zϩ or ZϪ rat agrin muscles was striking, supporting the notion that ACh is an AChR (13) or membrane-anchored cyan fluorescent protein (as con- declustering factor in vivo. trols) were added 24–36 h before CCh treatment. After fixation, Because AChR aggregates form and persist on uninnervated cultures were stained with rat anti-AChR (mAb35; Sigma) and myotubes in vivo (5, 6) and in vitro (2), we considered the mouse anti-rat agrin (mAb131; Stressgen Biotechnologies, San possibility that axons lacking both agrin and ACh failed to Diego) and evaluated blindly. innervate muscles. However, dko axons formed nerve trunks that Methods for preparing nerve–muscle cocultures were modi- entered muscles, branched, and terminated on myotubes; exu- fied from published protocols (14, 15) as detailed in Supporting berant growth already documented in chatϪ/Ϫ and agrnϪ/Ϫ Text, which is published as supporting information on the PNAS mutants (3, 10) was also present in dko mice (Figs. 1 b and c and web site. Motor neurons from one to two agrnϪ/Ϫ or agrnϩ/Ϫ 7).