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Changes in the Distribution of Synapse Specific Molecules at the Neuromuscular Junction upon Synaptic Retraction
CHANGES IN THE DISTRIBUTION OF SYNAPSE SPECIFIC MOLECULES AT THE NEUROMUSCULAR JUNCTION UPON SYNAPTIC RETRACTION
Michael J.Werle
Abstract.. ... . . . . .. . .. . .. ... . .. ... . .. ... . .. ... ... . .. . .. . .. . .. . . . . 56 I. INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 11. SYNAPTICRETRACTION. ... ... . .. ... ... ... ... . . . . .. . .. ... . .. . .. . 57 111. ACETYLCHOLINESTERASE . ... . .. ... . . . ... . . . ... ... ... ... . .. . .. . 59 IV. ACETYLCHOLINERECEETORS... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 V. AGRIN.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 VI. PRESYNAPTICMOLECULES...................................... 61 VII. SYNAPSE SPECIFIC CARBOHYDRATES. . . . . . . . . . . . . . . . . . . . . . . . . . . 62 VIII. OTHER SYNAPSE SPECIHC MOLECULES. . . . . . . . . . . . . . . . . . . . . . . . . 63 IX. CONCLUSIONS.. . . . . . , . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 63 References. .. .. . . . . . .. . . . . . . . .. . . _ .. . . . . . .. . . _... . . . . . . . . . . . . . . . . 63
Advances in Oqpn Bidogy Volume 2,pages 55-66. Copyright 0 1997 by JAI Presp Inc All rights of reproductionin any form reserved. ISBN: 0-7623-0222-4
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ABSTRACT Synaptic retraction occurs when the presynaptic nerve terminal withdraws from the postsynaptic structure. This process occurs both during development where it is especially prevalent during the process of synapse elimination, and throughout life as synapses change shape and size during synaptic remodeling. While much is known about the cellular mechanisms that control the formation of the synapse, relatively little is known about the cellular mechanisms that function during the retraction of a synapse. The smallest unit of the synapse must be the molecules that together form that cellular structure. Thus, to understand the formation of the synapse one must understand how those molecules come to be located at the synapse, while to understand synaptic retraction one must know how or if those molecules are removed from the synapse. In this article I will consider changes in the distribution of acetylcholinesterase, acetylcholine receptors, agrin, presynaptic molecules, synapse specific carbohydrates and other synapse specific molecules during synaptic retraction. By determining how these molecules are controlled during synapse formation and retraction, the basic mechanisms that control synapse formation and elimination may be determined.
1.
INTRODUCTION
During development, processes from the motor neuron grow out from the spinal cord and contact the developing myotubes. Exactly at the point of nerve-muscle contact functional synaptic specializations are formed (for a review see Hall and Sanes, 1993). In the nerve terminal the structural aspects of these specializations are called the presynaptic apparatus, and include the aggregation of vesicles that contain the neurotransmitter acetylcholine, and the formation of an electron dense aggregation of proteins on the surface of the nerve terminal called the active zone. Synaptic vesicles fuse with the plasma membrane of the nerve terminal at the active zone releasing their contents into the synaptic cleft. In and on the surface of the muscle fiber the synaptic specializations are called the postsynaptic apparatus, and include the aggregation of the receptor for the neurotransmitter acetylcholine (Anderson and Cohen, 1977),as well as aggregates of a 43kD protein named rapsyn (Froehner et al., 1981; Appel et al., 1995), a 58 kD protein associated with the dystroglycan complex named syntrophin (Froehneret al., 1987;Adams et al., 1993; Peters et al., 1994), a-actinin (Bloch and Hall, 1983; Shadiak and Nitkin, 1991), vinculin (Bloch and Hall, 1983; Shadiak and Nitkin, 1991), and the dystroglycan complex (Sugiyame et al., 1994,Cohen et al., 1995).Surrounding the muscle fiber, and the Schwann cell which caps the nerve terminal, is a basal lamina which contains molecules of collagen, heparin, nidogen, and laminin. The basal lamina extends into and occupies the space between the nerve terminal and muscle fiber, and this portion of the basal lamina is referred to as the synaptic basal lamina. In addition to the typical proteins found in basal lamina, synaptic basal lamina
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contains high quantities of acetylcholinesterase(McMahan et al., 1978; Anglister et al., 1994), heparin sulfate proteoglycans (Anderson and Fambrough, 1983), laminin p2 (s-laminin) (Hunter et al., 1989; Martin et al., 1995 ), collagen (type IV) a 3 and a4 chains, laminin A (Sanes et al., 1990), a uniquely glycosylated entactin (Chiu and KO,1994),protease nexin I (Festoff et al., 198l), thrombospondin-4 (Arber and Caroni, 1995) and agrin (Reist et al., 1987). Concurrentwith the development of synaptic specializationsis the developmental process by which each synaptic site is contacted by a superfluous number of neurites and, as development of the synapse proceeds, the number of neuronal inputs at that synaptic site is reduced until the adult pattern of innervation is achieved (Redfern, 1970; Brown et a1.,1976). This reduction in the number of neuronal inputs is referred to as synapse elimination. Synapse elimination occurs via competition between inputs at the same synaptic site (reviewed in Betz et al., 1990; Herrera and Werle, 1990). The amount of synaptic activity greatly affects the rate of synapse elimination, with increased activity increasing the rate of synapse elimination, and decreased activity decreasing the rate of synapse elimination (reviewed in Thompson, 1985). Another factor in the competition between neurons for the same synaptic space is the size of the motor unit from which the terminal originates. Terminals from active motor units are at a competitive advantage when competing with terminals from inactive motor units (Ridge and Betz, 1984 ) but see Callaway et al., (1987). While much is known about the process of synapse elimination, the cellular and molecular mechanisms that control synapse elimination are poorly understood. If one defines synaptogenesis as the process by which mature, stabilized synapses are formed, then synapse elimination is an integral part of synaptogenesis, and the cellular and molecular mechanisms that control the formation of synaptic specializations are likely to be involved in the mechanisms that control synapse elimination.
II.
SYNAPTIC RETRACTION
The retraction or elimination of synapses occurs during normal development and also in many neurodegenerative diseases. In this article I will be focusing on what is known about the distribution of synapse specific molecules during synaptic retraction. Many molecules have been identified that are aggregated during the formation of synapses; however, what happens to those molecules during the removal of the synapse has been less well characterized. In particular, the neuromuscular junction has proven to be a very useful model synapse to study synapse formation, and it is probable that this synapse will be equally useful in the study of synaptic retraction. As seen in Figure 1, the frog neuromuscularjunction is an exquisite preparation for studying fine details of synaptic morphology. The nerve terminals are well separated on the muscle fiber, thus individual nerve terminal branches can be readily identified. The nerve terminal in Figure 1
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Figure 7. Frog neuromuscular junction stained by the Karnovsky method to reveal the distribution of acetylcholinesterase (Top panel) and rhodamine conjugated a-bungarotoxin binding to acetylcholine receptors (bottom panel). The open arrow in the top panel indicatesan area on the muscle fiber that stains positively for cholinesterase yet lacks a nerve terminal over that area; this area is referred to as an empty synaptic' gutter. The solid arrow in the bottom panel indicatesthat the empty synapticgutter lacks acetylcholine receptors. The scale bar is 70 Tm.
illustrates the classic appearance of a nerve terminal that has retracted. The distal tip of the nerve terminal lies in the synaptic gutter, and distal to the nerve terminal one can see cholinesterasereaction product in acharacteristic pattern of aggregation in the empty junctional folds. The area marked with the star was a synapse at a prior time; however, the nerve terminal has retracted, and the former synaptic site is no longer functioning. When the same endplate is viewed under fluorescent optics to reveal the distribution of rhodamine a-bungarotoxin bound to the acetylcholine receptors it is clear that while acetylcholinesterase is present in the abandoned gutter the acetylcholine receptor has been removed. Thus, in this example we can see that the molecular components of the synapse are removed at different rates when a nerve terminal retracts. We will now consider what happens to these, and other molecules when a nerve terminal retracts.
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ACETYLCHOLINESTERASE (AChE)
The most stable and persistent molecule at the synapse is probably the cholinesterase. It has been reported that cholinesterase will remain at abandoned synaptic gutters for up to two years (Krause and Wernig, 1985). Presumably, because of this fact and the fact that a histological marker for cholinesterase has been available for over 30 years (Katnovsky and Roots, 1964), this enzyme has been the best characterized molecule at abandoned synaptic sites. Based on static images of histologically stained endplates it was hypothesized that the empty synaptic gutters were synaptic sites abandoned by the nerve terminal (Wernig et al., 1981). These empty gutters were more prevalent in the summer than in the winter leading to speculation that there is constantremodelingof the neuromuscular junction based on synaptic activity (Wernig et al., 1980). Indeed, repeated in vivo observationsof the frog neuromuscularjunction have revealed the neuromuscular junction to be highly plastic with nerve terminals growing and retracting within time frames of days to weeks (Herrera et al., 1990; Langenfeld-Oster et al., 1993; Chen and KO, 1994). Thus, the deposition of cholinesterase into synaptic basal lamina is very stable, and this remnant cholinesterase is an excellent marker for abandoned synaptic sites.
IV. ACETYLCHOLINE RECEPTORS (AChR) The distribution of the AChRs has also been well studied in synapse formation and elimination.The distribution of the AChR has been immensely aided by the use of the snake venom a-bungarotoxin. The binding of a-bungarotoxin to the AChR blocks its ability to bind acetyl choline, and thus blocks synaptic transmission. Conjugating fluorescent markers to the a-bungarotoxin provides a powerful method of visualizing the location of the AChRs (Figure 1 bottom panel). In static views of the endplate it has been noted that AChRs are absent from empty synaptic gutters, thus, even though AChE is still present, the AChRs have dispersed (Wernig et al., 1981). Thus, the time course of the removal of synapse specific molecules is different, AChRs are absent at a time when AChE is present. While this seems to be a rather simplistic statement it underlies a basic need to understand the time course of the removal of other synapse specific molecules. Understanding the removal of the molecules will aid in understanding the mechanism(s) that drive the dispersal of the synaptic apparatus, just as knowing the time course of the aggregation of the synapse specific molecules has aided the understanding of the mechanisms that control synapse formation. The distribution of AChRs at denervated synaptic sites differs from that at normal muscle followingretraction. At endplatesof denervated muscles the AChRs will remain in the abandoned synaptic sites for up to 2 months (Reist et al.. 1987). This is an interesting observation, and may reveal much about the mechanisms that control the stabilization of the postsynaptic apparatus. On the one hand, when a
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nerve terminal retracts in a normal muscle, AChRs are no longer found in the a6andoned gutter, while when a nerve terminal is removed following nerve crush the AChRs will remain. Thus, an empty gutter will either have or not have AChRs, depending on how the nerve that originally innervated that gutter was removed, or by the level of activity of the neuromuscular system. One interesting difference between these two situationsis that during normal synapticretraction synaptic and muscle activity are preserved, while in the case of denervation synaptic and muscle activity are lost. Thus, the dispersal of AChRs could be directed by an activity dependent process. In vivo observations have revealed a great deal about the dynamics of the synapse during synaptic retraction. Lichtman and colleagues have revealed that prior to the retraction of a nerve terminal, the AChRs under the nerve terminal disperse (Balice-Gordon and Lichtman, 1993). Further, if a-bungarotoxin is puffed onto a portion of a neuromuscular junction, the AChRs (both blocked and unblocked) in that portion of the synapse that is blocked by the toxin will disperse, followed by retraction of the nerve terminal (Balice Gordon and Lichtmann, 1994). Thus, functional synaptic transmission is essential to maintain the synaptic apparatus.
V. ACRIN Signals that control the formation of the synapse are exchanged between the muscle and nerve, and many lines of evidence suggest that agrin is involved in the formation of the postsynaptic apparatus during development, maintenance of the apparatus in the adult, and reformation of the postsynaptic apparatus during regeneration (reviewed in McMahan, 1990;McMahan et al., 1992). Recent experiments have revealed information on the molecular nature of agrin. Anti-agrin antibodies block the nerve induced aggregation of AChRs in nerve-muscle cocultures (Reist et al., 1992). The gene for agrin has been cloned in rat (Rupp et al., 1991) chick (Tsim et al., 1992), and ray (Smith et al., 1992). There is one gene that codes for agrin, and, via alternative splicing, different isoforms of agrin are produced (Ruegg et al., 1992; Ferns et al., 1992; Ferns et al., 1993). In chick, these isoforms vary greatly in their ability to direct the aggregation of AChRs on the surface of cultured chick myotubes (Ruegg et al., 1992; Gesemann et al., 1995, 1996), dependent on alternative splicing at two sites, designated region A and region B. In order for agrin to be active in AChR aggregation, there must be an insert at both region A and region B. PCR studies and oligo in situ hybridizations reveal that motor neurons produce agrin isoforms having inserts at both region A and B, while muscle fibers and Schwann cells produce agrin isoforms that lack an insert at Region B (Ma et al., 1994 ). Therefore, motor neurons produce an isoform of agrin that is capable of directing the aggregation of AChRs on cultured chick myotubes, while muscle fibers and Schwann cells make an isoform of agrin that is inactive. Further, there is another alternatively spliced location of agrin at the
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N-terminus near the putative signal sequence of agrin that is also differentially express'ed in neurons and non-neurons (Tsen et al., 1995) and this region is required for agrin to bind to extracellular matrix @enzer et al., 1995). A fourth region of the agrin molecule has been identified that is required in order for agrin to bind to isolated preparations of chick retina basal lamina (Stephan Kroger, personal communication) and recent experiments have also revealed a fifth separate region of the agrin molecule that binds to the dystroglycan complex (Bowe et al., 1994; Campanelli et al., 1994; Sugiyama et al., 1994; Sealock and Froehner 1994; Gee et al., 1994). Thus, there are several domains on the agrin molecule which either bind or interact with other synaptic components. Completely understanding the linkage between basal lamina binding and the ability of agrin to induce and maintain the postsynaptic apparatus will be critical to understanding the complete function of agrin during synapse formation, elimination and remodeling. When a nerve is denervated, agrin remains stably bound to synaptic basal lamina, even after the nerve and Schwann cell have abandoned the synaptic gutter (Reist et al., 1987). Not only does agrin remain stably bound to the basal lamina, it retains its AChR aggregating activity even after myotube degeneration and regeneration (Burden et al., 1979; McMahan and Slater, 1984). Thus, when a muscle is denervated the agrin maintains the postsynaptic apparatus until the nerve terminal regenerates and reoccupies the old synaptic site. However, in contrast to experimental denervation, it is interesting to note that during normal synaptic retraction the AChRs under the nerve terminal are quickly removed, and the agrin that was stably bound to the basal lamina that induced the aggregation of those AChRs must have been either blocked or removed. When normal frog neuromuscular junctions are stained with antiagrin antibodies, abandoned synaptic gutters lack agrin immunoreactivity. Therefore, the agrin that was once bound to the synaptic basal lamina has been removed. This seems to be an interesting paradox, if a nerve terminal retracts, ths agrin is removed, however, if the nerve is crushed so that the entire nerve terminal is removed the agrin persists. Thus, there appears to be a selective mechanism by which agrin can be removed from the synaptic basal lamina. One obvious difference between synaptic retraction in a normal muscle, and the removal of synapses following nerve crush is that during synaptic retraction in normal muscle the synapse and the muscle fiber remain active, while in the case of denervation, neuromuscular activity is blocked. It will be interesting to determine whether activity plays a role in the removal of agrin from synaptic basal lamina, and what role the removal of agrin plays in synaptic remodelling.
VI.
PRESYNAPTIC MOLECULES
Obviously, when a nerve terminal retracts, all of the presynaptic specializationsare lost. The time course of the changes in the presynaptic organization will be difficult
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to study. In a freeze fracture study, it was found that the distal tips of some nerve tkrminals contained active zones that were disorganized in their structure(Pumplin, 1983). It was impossible from these studies to determine whether a given nerve terminal was in the process of elongation or retraction, but one would expect that the disruption of the active zone would be one of the first signs of synaptic retraction. Recent experiments have been directed towards identifyingthe molecular signals which trigger the differentiationof the presynaptic apparatus (Werle and McMahan, 1991; Porter et al., 1995; Dai and Peng, 1995). Sanes and colleagues (1995) have shown that when cultured neurons are grown on s-laminin, or simply the tripeptide leu-arg-glu @RE) contained within the s-laminin molecule, the neurons stop growing and show signs of presynaptic specializations.Further, a 16 peptide C-terminal fragment containing the LRE tripeptide is necessary for synaptic localization of s-laminin (Martin et al., 1995). While basic fibroblast growth factor (FGF)has been shown to induce changes in the level of calcium in neurons (Dai and Peng. 1995), the exact molecule that induces the presynaptic specialization has yet to be identified, and the mechanism by which it exerts its effect on the nerve terminal has yet to be described. However, experiments provide strong evidence that a molecule stably bound to synaptic basal lamina is capable of directing the formation of the presynaptic apparatus (Sanes et al., 1978; Glicksman and Sanes, 1983). Once the identity of the presynaptic inducing element is determined it will be of great interest to also determine whether this basal lamina molecule is removed from the synaptic basal lamina. It is highly likely that the removal of this signal would then lead to synaptic retraction.
VII. SYNAPSE SPECIFIC CARBOHYDRATES An interesting feature about synapses is that not only do they contain synapse specific isoforms based on their amino acid sequence, but they also contain isoforms that are synapse specific based on their glycosylation. For example, entactin, found throughout the basal lamina of the muscle is differentially glycosylated (Chiu and KO,1994) in the synaptic basal lamina. It is not known what role this modificationof the entactinplays in the synapticbasal lamina. The mechanism underlying this specific distribution of carbohydrates may be linked to the specific distribution of cytoplasmic enzymes which glycosylate. For example, the enzyme N-acetylgalctosaminyl transferase is localized to the sub-synaptic cytoplasm in muscle (Scott et al., 1990) Analysis of the distribution of synapse specific carbohydrates have been greatly aided by the use of lectins that bind specific sugars. For example the lectins peanut agglutinin (KO1987), Dolichos biflorus agglutinin,and Vicia villosa-34 agglutinin,(WA,Scott et al., 1988) have all been shown to bind to sugars that are concentrated at the neuromuscular junction. The role of these sugars in synaptic structure and function has yet to be determined but it has been found that treatment of myotube cultures with the lectin VVA will inhibit agrin induced aggregation of the postsynaptic apparatus (Martin and Sanes, 1995).
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OTHER SYNAPSE SPECIFIC MOLECULES
Approximately 50 synapse associated molecules have been identified (see introduction; Hall and Sanes, 1993). While it has been shown that these molecules are aggregated at neuromuscularjunctions, it has yet to be determined when and if they are removed following synaptic retraction.
IX. CONCLUSIONS Synapses are highly dynamic structures during development and throughout life. The formation and removal of the synapse is a very precise process, and understanding the cellular and molecular mechanisms controlling the formation and retraction of synaptic structures will be of great importance. Many molecules are specifically concentrated at synapses, and several are modified such that a specialized form of the molecule is synapse specific. Current research has primarily focused on the mechanismsthat control the formation of the synapse; however, the mechanisms that control the breakup of these synapses are poorly understood. It will be interesting to determine whether molecules that are aggregated at different times and rates during synapse formation are dispersed in complementarypatterns. Thus, it may be that the same basic mechanisms that control the formation of the synapse are also involved in the retraction and elimination of that synapse.
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Hall, Z.W., & Sanes, J.R. (1993). Synaptic structure and development: the neuromuscular junction. Cell 72 (Suppl) 99-121. Herrera, A.A., Banner, L.R., & Nagaya, N. (1990)Repeated,in vivo observationof frog neuromuscular junctions: remodelling involves concurrent growth and retraction. J. Neurocytol. 19(1), 85-99. Herrera, A.A., & Werle, M.J. (1990).Mechanismsof elimination, remodeling,and competition at frog neuromuscularjunctions. J, Neurobiol. 21(1), 73-98. Hunter, D.D., Shah, V.,Merlie, J.P., & Sanes,J.R. (1989). A laminin-likeadhesiveprotein concentrated in the synaptic cleft of the neuromuscularjunction. Nature 338(6212), 229-234. Karnovsky, M.J., & Roots, L. (1964). A “dimt-coloring” thiocholine method for cholinesterases. J. Histochem. and Cytochem. 12,219-221. KO, C.P. (1987). A lectin, peanut agglutinin, as a probe for the extracellular matrix in living neuromuscularjunctions. J. Neurocytol. 16(4), 567-576. Krause, M., & Wernig, A. (1985). The distribution of acetylcholine receptors in the n o d and denervated neuromuscularjunction of the frog. J. Neurocytol. 14(5), 765-780. Langenfeld-Oster,B., Dorlochter,M., & Wemig, A. (1993). Regularand photodamage,enhancedremodelling in vitally stained frog and mouse neuromuscular junctions. J. Neumcytol. 22(7), 517-530. Ma, E., Morgan, R., &Godkey, E.W. (1994).Distributionofagrin mRNAs in the chickembryonervous system. J. Neurosci. 14(5), 2943-2952. Martin, P.T., & Sanes, J.R. (1995).Role for asynapse, specificcarbohydrateinagrin, induced clustering of acetylcholine receptors. Neuron. (4). 743-754. Martin, P.T., Ettinger, A.J., & Sanes, J.R. (1995). A synaptic localization domain in the synaptic cleft protein laminin beta 2 (s-laminin) [see comments] Science 269(5222), 413-416 [Comment in: Science 269(5222):362-3631. McMahan, U.J., Sanes, J.R., & Marshall, L.M. (1978). Cholinesterase is associated with the basal lamina at the neuromuscularjunction. Nature 271(5641), 172-174. McMahan, U.J., & Slater, C.R. (1984). The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle. J. Cell Biol. 98(4), 1453-1473. McMahan, U.J. (1990). The agrin hypothesis. Cold Spring Harb. Symp. Quant. Biol. 55,407-418. McMahan, U.J., Horton, S.E., Werle, M.J., Honig, L.S., Kroger, S., Ruegg, M.A., &Escher, G. (1992). Agrin isoforms and their role in synaptogenesis.Cum. Opin. Cell.Biol. 4(5), 869-874. Peters, M.F., Kramarcy, N.R., Sealock, R., & Froehner, S.C. (1994). beta 2, Syntrophin: localization at the neuromuscularjunctionin skeletal muscle. Neuroreport 5(13), 1577-1580. Porter, B.E., Weis, J., & Sanes, J.R. (1995).A motoneuron-selectivestop signal in the synaptic protein s-laminin. Neuron 14(3), 549-559. Purnplin,D.W. (1983). Normal variations in presynapticactive zones of frog neuromuscularjunctions. J. Neurocytol. 12(2), 317-323. Redfern, P.A. (1970). Neuromusculartransmission in new-born rats. J. Physiol. 209,701-709. Reist, N.E., Magill, C., & McMahan, U.J. (1987). Agrin, l i e molecules at synaptic sites in normal, denervated, and damaged skeletal muscles. J. Cell Biol. 105(6),2457-2469. Reist, N.E., Werle, M.J., & McMahan, U.J. (1992). Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscularjunctions. Neuron 8(5), 865-868. Ridge, R.M., & Betz, W.J. (1984). The effect of selective, chronic stimulation on motor unit size in developing rat muscle. J. Neurosci. 4( lo), 2614-2620. Ruegg, M.A., Tsim, K.W., Horton, S.E., Kroger, S., Escher, G., Gensch, E.M., & McMahan, U.J. (1992). The agrin gene codes for a family of basal lamina proteins that differ in function and distribution.Neuron 8(4), 691-699. Rupp, F., Payan, D.G., Magill-Solc, C., Cowan, D.M., & Scheller, R.H. (1991). Structure and expression of a rat agrin. Neuron 6(5), 81 1-823. Sanes, J.R., Marshall, L.M., & McMahan, U.J. (1978).Reinnervationof muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J. Cell Biol. 78(1), 176-198.
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Sanes, J.R., Engvall, E.,Butkowski, R., & Hunter, D.D. (1990). Molecular heterogeneity of basal laminae.: isofonns of laminin and collagen N at the neuromuscular junction and elsewhere. J. Cell Biol. 111(4), 1685-1699. Scott, L.J., Bacou, F., & Sanes, J.R. (1988). A synapse, specific carbohydrate at the neuromuscular junction: associationwith both acetylcholinesteraseand aglycolipid. J. Neurosci. 8(3), 932-944. Scott, L.J., Balsamo, J., Sanes, J.R., & Lilien, J. (1990). Synapticlocalizationand neural regulation of an N-acetylgalactosaminyl transferasein skeletal muscle. J. Neurosci. 10(1), 346-350. Sealock,R., & Froehner, S.C. (1994). Dystrophin, associated proteins and synapse formation: is alpha, dystroglycan the agrin receptor? Cell 77(5), 617-619. Shdiack, A.M., & Nitkin, R.M. (1991). Agrin induces alpha, actinin, filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes. J. Neurobiol. 22(6), 617-628. Smith, M. A., Magill, Solc, C., Rupp, F.,Yao, Y.-M., Schilling, J.W., Snow, P., & McMahan, U.J. (1992). Isolation and characterization of an agrin homolog in the marine ray. Mol. Cell. Neurosci. 3,406-417. Sugiyama,J., Bowen, D.C., &Hall, Z.W. (1994). Dystroglycan binds nerve and muscle agrin. Neuron. 13(1), 103-115.
Thompson, W.(1985), Activity and synapseeliminationat the neuromuscular junction. Cell. and Mol. Neurol. 5,167-182. Tsen, G., Napier, A., Halfter, W., & Cole, G.J. (1995). Identification of a novel alternatively spliced agrin mRNA that is preferentially expressed in non-neuronal cells. J. Biol. Chem. 270(27), 15934-15937.
Tsim, K.W., Ruegg, M.A., Escher, G., Kroger, S., & McMahan, U.J. (1992). cDNA that encodes active agrin. Neuron 8(4), 677-689. [published erratum appears in Neuron 9(2):following3811 Wernig, A., Pecot-Dechavassine, M., &Stover, H. (1980). Sprouting and regressionof the nerve at the frog neuromuscular junction in normal conditionsand after prolonged paralysis with curare. J. Neumytol. 9(3), 278-303. Wernig, A., A n d , A.P., Bieser, A., & Schwarz, U. (1981). Abandoned synaptic sites in muscles of normal adult frog. Neurosci, Lett. 23(2), 105-110. Werle, M.J., & McMahan, U.J. (1991). Molecules that induce the formation of synaptic apparatus. Restorative Neurol. 5,269-273.
Michael J.Werle
Abstract.. ... . . . . .. . .. . .. ... . .. ... . .. ... . .. ... ... . .. . .. . .. . .. . . . . 56 I. INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 11. SYNAPTICRETRACTION. ... ... . .. ... ... ... ... . . . . .. . .. ... . .. . .. . 57 111. ACETYLCHOLINESTERASE . ... . .. ... . . . ... . . . ... ... ... ... . .. . .. . 59 IV. ACETYLCHOLINERECEETORS... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 V. AGRIN.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 VI. PRESYNAPTICMOLECULES...................................... 61 VII. SYNAPSE SPECIFIC CARBOHYDRATES. . . . . . . . . . . . . . . . . . . . . . . . . . . 62 VIII. OTHER SYNAPSE SPECIHC MOLECULES. . . . . . . . . . . . . . . . . . . . . . . . . 63 IX. CONCLUSIONS.. . . . . . , . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 63 References. .. .. . . . . . .. . . . . . . . .. . . _ .. . . . . . .. . . _... . . . . . . . . . . . . . . . . 63
Advances in Oqpn Bidogy Volume 2,pages 55-66. Copyright 0 1997 by JAI Presp Inc All rights of reproductionin any form reserved. ISBN: 0-7623-0222-4
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ABSTRACT Synaptic retraction occurs when the presynaptic nerve terminal withdraws from the postsynaptic structure. This process occurs both during development where it is especially prevalent during the process of synapse elimination, and throughout life as synapses change shape and size during synaptic remodeling. While much is known about the cellular mechanisms that control the formation of the synapse, relatively little is known about the cellular mechanisms that function during the retraction of a synapse. The smallest unit of the synapse must be the molecules that together form that cellular structure. Thus, to understand the formation of the synapse one must understand how those molecules come to be located at the synapse, while to understand synaptic retraction one must know how or if those molecules are removed from the synapse. In this article I will consider changes in the distribution of acetylcholinesterase, acetylcholine receptors, agrin, presynaptic molecules, synapse specific carbohydrates and other synapse specific molecules during synaptic retraction. By determining how these molecules are controlled during synapse formation and retraction, the basic mechanisms that control synapse formation and elimination may be determined.
1.
INTRODUCTION
During development, processes from the motor neuron grow out from the spinal cord and contact the developing myotubes. Exactly at the point of nerve-muscle contact functional synaptic specializations are formed (for a review see Hall and Sanes, 1993). In the nerve terminal the structural aspects of these specializations are called the presynaptic apparatus, and include the aggregation of vesicles that contain the neurotransmitter acetylcholine, and the formation of an electron dense aggregation of proteins on the surface of the nerve terminal called the active zone. Synaptic vesicles fuse with the plasma membrane of the nerve terminal at the active zone releasing their contents into the synaptic cleft. In and on the surface of the muscle fiber the synaptic specializations are called the postsynaptic apparatus, and include the aggregation of the receptor for the neurotransmitter acetylcholine (Anderson and Cohen, 1977),as well as aggregates of a 43kD protein named rapsyn (Froehner et al., 1981; Appel et al., 1995), a 58 kD protein associated with the dystroglycan complex named syntrophin (Froehneret al., 1987;Adams et al., 1993; Peters et al., 1994), a-actinin (Bloch and Hall, 1983; Shadiak and Nitkin, 1991), vinculin (Bloch and Hall, 1983; Shadiak and Nitkin, 1991), and the dystroglycan complex (Sugiyame et al., 1994,Cohen et al., 1995).Surrounding the muscle fiber, and the Schwann cell which caps the nerve terminal, is a basal lamina which contains molecules of collagen, heparin, nidogen, and laminin. The basal lamina extends into and occupies the space between the nerve terminal and muscle fiber, and this portion of the basal lamina is referred to as the synaptic basal lamina. In addition to the typical proteins found in basal lamina, synaptic basal lamina
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contains high quantities of acetylcholinesterase(McMahan et al., 1978; Anglister et al., 1994), heparin sulfate proteoglycans (Anderson and Fambrough, 1983), laminin p2 (s-laminin) (Hunter et al., 1989; Martin et al., 1995 ), collagen (type IV) a 3 and a4 chains, laminin A (Sanes et al., 1990), a uniquely glycosylated entactin (Chiu and KO,1994),protease nexin I (Festoff et al., 198l), thrombospondin-4 (Arber and Caroni, 1995) and agrin (Reist et al., 1987). Concurrentwith the development of synaptic specializationsis the developmental process by which each synaptic site is contacted by a superfluous number of neurites and, as development of the synapse proceeds, the number of neuronal inputs at that synaptic site is reduced until the adult pattern of innervation is achieved (Redfern, 1970; Brown et a1.,1976). This reduction in the number of neuronal inputs is referred to as synapse elimination. Synapse elimination occurs via competition between inputs at the same synaptic site (reviewed in Betz et al., 1990; Herrera and Werle, 1990). The amount of synaptic activity greatly affects the rate of synapse elimination, with increased activity increasing the rate of synapse elimination, and decreased activity decreasing the rate of synapse elimination (reviewed in Thompson, 1985). Another factor in the competition between neurons for the same synaptic space is the size of the motor unit from which the terminal originates. Terminals from active motor units are at a competitive advantage when competing with terminals from inactive motor units (Ridge and Betz, 1984 ) but see Callaway et al., (1987). While much is known about the process of synapse elimination, the cellular and molecular mechanisms that control synapse elimination are poorly understood. If one defines synaptogenesis as the process by which mature, stabilized synapses are formed, then synapse elimination is an integral part of synaptogenesis, and the cellular and molecular mechanisms that control the formation of synaptic specializations are likely to be involved in the mechanisms that control synapse elimination.
II.
SYNAPTIC RETRACTION
The retraction or elimination of synapses occurs during normal development and also in many neurodegenerative diseases. In this article I will be focusing on what is known about the distribution of synapse specific molecules during synaptic retraction. Many molecules have been identified that are aggregated during the formation of synapses; however, what happens to those molecules during the removal of the synapse has been less well characterized. In particular, the neuromuscular junction has proven to be a very useful model synapse to study synapse formation, and it is probable that this synapse will be equally useful in the study of synaptic retraction. As seen in Figure 1, the frog neuromuscularjunction is an exquisite preparation for studying fine details of synaptic morphology. The nerve terminals are well separated on the muscle fiber, thus individual nerve terminal branches can be readily identified. The nerve terminal in Figure 1
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Figure 7. Frog neuromuscular junction stained by the Karnovsky method to reveal the distribution of acetylcholinesterase (Top panel) and rhodamine conjugated a-bungarotoxin binding to acetylcholine receptors (bottom panel). The open arrow in the top panel indicatesan area on the muscle fiber that stains positively for cholinesterase yet lacks a nerve terminal over that area; this area is referred to as an empty synaptic' gutter. The solid arrow in the bottom panel indicatesthat the empty synapticgutter lacks acetylcholine receptors. The scale bar is 70 Tm.
illustrates the classic appearance of a nerve terminal that has retracted. The distal tip of the nerve terminal lies in the synaptic gutter, and distal to the nerve terminal one can see cholinesterasereaction product in acharacteristic pattern of aggregation in the empty junctional folds. The area marked with the star was a synapse at a prior time; however, the nerve terminal has retracted, and the former synaptic site is no longer functioning. When the same endplate is viewed under fluorescent optics to reveal the distribution of rhodamine a-bungarotoxin bound to the acetylcholine receptors it is clear that while acetylcholinesterase is present in the abandoned gutter the acetylcholine receptor has been removed. Thus, in this example we can see that the molecular components of the synapse are removed at different rates when a nerve terminal retracts. We will now consider what happens to these, and other molecules when a nerve terminal retracts.
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ACETYLCHOLINESTERASE (AChE)
The most stable and persistent molecule at the synapse is probably the cholinesterase. It has been reported that cholinesterase will remain at abandoned synaptic gutters for up to two years (Krause and Wernig, 1985). Presumably, because of this fact and the fact that a histological marker for cholinesterase has been available for over 30 years (Katnovsky and Roots, 1964), this enzyme has been the best characterized molecule at abandoned synaptic sites. Based on static images of histologically stained endplates it was hypothesized that the empty synaptic gutters were synaptic sites abandoned by the nerve terminal (Wernig et al., 1981). These empty gutters were more prevalent in the summer than in the winter leading to speculation that there is constantremodelingof the neuromuscular junction based on synaptic activity (Wernig et al., 1980). Indeed, repeated in vivo observationsof the frog neuromuscularjunction have revealed the neuromuscular junction to be highly plastic with nerve terminals growing and retracting within time frames of days to weeks (Herrera et al., 1990; Langenfeld-Oster et al., 1993; Chen and KO, 1994). Thus, the deposition of cholinesterase into synaptic basal lamina is very stable, and this remnant cholinesterase is an excellent marker for abandoned synaptic sites.
IV. ACETYLCHOLINE RECEPTORS (AChR) The distribution of the AChRs has also been well studied in synapse formation and elimination.The distribution of the AChR has been immensely aided by the use of the snake venom a-bungarotoxin. The binding of a-bungarotoxin to the AChR blocks its ability to bind acetyl choline, and thus blocks synaptic transmission. Conjugating fluorescent markers to the a-bungarotoxin provides a powerful method of visualizing the location of the AChRs (Figure 1 bottom panel). In static views of the endplate it has been noted that AChRs are absent from empty synaptic gutters, thus, even though AChE is still present, the AChRs have dispersed (Wernig et al., 1981). Thus, the time course of the removal of synapse specific molecules is different, AChRs are absent at a time when AChE is present. While this seems to be a rather simplistic statement it underlies a basic need to understand the time course of the removal of other synapse specific molecules. Understanding the removal of the molecules will aid in understanding the mechanism(s) that drive the dispersal of the synaptic apparatus, just as knowing the time course of the aggregation of the synapse specific molecules has aided the understanding of the mechanisms that control synapse formation. The distribution of AChRs at denervated synaptic sites differs from that at normal muscle followingretraction. At endplatesof denervated muscles the AChRs will remain in the abandoned synaptic sites for up to 2 months (Reist et al.. 1987). This is an interesting observation, and may reveal much about the mechanisms that control the stabilization of the postsynaptic apparatus. On the one hand, when a
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nerve terminal retracts in a normal muscle, AChRs are no longer found in the a6andoned gutter, while when a nerve terminal is removed following nerve crush the AChRs will remain. Thus, an empty gutter will either have or not have AChRs, depending on how the nerve that originally innervated that gutter was removed, or by the level of activity of the neuromuscular system. One interesting difference between these two situationsis that during normal synapticretraction synaptic and muscle activity are preserved, while in the case of denervation synaptic and muscle activity are lost. Thus, the dispersal of AChRs could be directed by an activity dependent process. In vivo observations have revealed a great deal about the dynamics of the synapse during synaptic retraction. Lichtman and colleagues have revealed that prior to the retraction of a nerve terminal, the AChRs under the nerve terminal disperse (Balice-Gordon and Lichtman, 1993). Further, if a-bungarotoxin is puffed onto a portion of a neuromuscular junction, the AChRs (both blocked and unblocked) in that portion of the synapse that is blocked by the toxin will disperse, followed by retraction of the nerve terminal (Balice Gordon and Lichtmann, 1994). Thus, functional synaptic transmission is essential to maintain the synaptic apparatus.
V. ACRIN Signals that control the formation of the synapse are exchanged between the muscle and nerve, and many lines of evidence suggest that agrin is involved in the formation of the postsynaptic apparatus during development, maintenance of the apparatus in the adult, and reformation of the postsynaptic apparatus during regeneration (reviewed in McMahan, 1990;McMahan et al., 1992). Recent experiments have revealed information on the molecular nature of agrin. Anti-agrin antibodies block the nerve induced aggregation of AChRs in nerve-muscle cocultures (Reist et al., 1992). The gene for agrin has been cloned in rat (Rupp et al., 1991) chick (Tsim et al., 1992), and ray (Smith et al., 1992). There is one gene that codes for agrin, and, via alternative splicing, different isoforms of agrin are produced (Ruegg et al., 1992; Ferns et al., 1992; Ferns et al., 1993). In chick, these isoforms vary greatly in their ability to direct the aggregation of AChRs on the surface of cultured chick myotubes (Ruegg et al., 1992; Gesemann et al., 1995, 1996), dependent on alternative splicing at two sites, designated region A and region B. In order for agrin to be active in AChR aggregation, there must be an insert at both region A and region B. PCR studies and oligo in situ hybridizations reveal that motor neurons produce agrin isoforms having inserts at both region A and B, while muscle fibers and Schwann cells produce agrin isoforms that lack an insert at Region B (Ma et al., 1994 ). Therefore, motor neurons produce an isoform of agrin that is capable of directing the aggregation of AChRs on cultured chick myotubes, while muscle fibers and Schwann cells make an isoform of agrin that is inactive. Further, there is another alternatively spliced location of agrin at the
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N-terminus near the putative signal sequence of agrin that is also differentially express'ed in neurons and non-neurons (Tsen et al., 1995) and this region is required for agrin to bind to extracellular matrix @enzer et al., 1995). A fourth region of the agrin molecule has been identified that is required in order for agrin to bind to isolated preparations of chick retina basal lamina (Stephan Kroger, personal communication) and recent experiments have also revealed a fifth separate region of the agrin molecule that binds to the dystroglycan complex (Bowe et al., 1994; Campanelli et al., 1994; Sugiyama et al., 1994; Sealock and Froehner 1994; Gee et al., 1994). Thus, there are several domains on the agrin molecule which either bind or interact with other synaptic components. Completely understanding the linkage between basal lamina binding and the ability of agrin to induce and maintain the postsynaptic apparatus will be critical to understanding the complete function of agrin during synapse formation, elimination and remodeling. When a nerve is denervated, agrin remains stably bound to synaptic basal lamina, even after the nerve and Schwann cell have abandoned the synaptic gutter (Reist et al., 1987). Not only does agrin remain stably bound to the basal lamina, it retains its AChR aggregating activity even after myotube degeneration and regeneration (Burden et al., 1979; McMahan and Slater, 1984). Thus, when a muscle is denervated the agrin maintains the postsynaptic apparatus until the nerve terminal regenerates and reoccupies the old synaptic site. However, in contrast to experimental denervation, it is interesting to note that during normal synaptic retraction the AChRs under the nerve terminal are quickly removed, and the agrin that was stably bound to the basal lamina that induced the aggregation of those AChRs must have been either blocked or removed. When normal frog neuromuscular junctions are stained with antiagrin antibodies, abandoned synaptic gutters lack agrin immunoreactivity. Therefore, the agrin that was once bound to the synaptic basal lamina has been removed. This seems to be an interesting paradox, if a nerve terminal retracts, ths agrin is removed, however, if the nerve is crushed so that the entire nerve terminal is removed the agrin persists. Thus, there appears to be a selective mechanism by which agrin can be removed from the synaptic basal lamina. One obvious difference between synaptic retraction in a normal muscle, and the removal of synapses following nerve crush is that during synaptic retraction in normal muscle the synapse and the muscle fiber remain active, while in the case of denervation, neuromuscular activity is blocked. It will be interesting to determine whether activity plays a role in the removal of agrin from synaptic basal lamina, and what role the removal of agrin plays in synaptic remodelling.
VI.
PRESYNAPTIC MOLECULES
Obviously, when a nerve terminal retracts, all of the presynaptic specializationsare lost. The time course of the changes in the presynaptic organization will be difficult
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to study. In a freeze fracture study, it was found that the distal tips of some nerve tkrminals contained active zones that were disorganized in their structure(Pumplin, 1983). It was impossible from these studies to determine whether a given nerve terminal was in the process of elongation or retraction, but one would expect that the disruption of the active zone would be one of the first signs of synaptic retraction. Recent experiments have been directed towards identifyingthe molecular signals which trigger the differentiationof the presynaptic apparatus (Werle and McMahan, 1991; Porter et al., 1995; Dai and Peng, 1995). Sanes and colleagues (1995) have shown that when cultured neurons are grown on s-laminin, or simply the tripeptide leu-arg-glu @RE) contained within the s-laminin molecule, the neurons stop growing and show signs of presynaptic specializations.Further, a 16 peptide C-terminal fragment containing the LRE tripeptide is necessary for synaptic localization of s-laminin (Martin et al., 1995). While basic fibroblast growth factor (FGF)has been shown to induce changes in the level of calcium in neurons (Dai and Peng. 1995), the exact molecule that induces the presynaptic specialization has yet to be identified, and the mechanism by which it exerts its effect on the nerve terminal has yet to be described. However, experiments provide strong evidence that a molecule stably bound to synaptic basal lamina is capable of directing the formation of the presynaptic apparatus (Sanes et al., 1978; Glicksman and Sanes, 1983). Once the identity of the presynaptic inducing element is determined it will be of great interest to also determine whether this basal lamina molecule is removed from the synaptic basal lamina. It is highly likely that the removal of this signal would then lead to synaptic retraction.
VII. SYNAPSE SPECIFIC CARBOHYDRATES An interesting feature about synapses is that not only do they contain synapse specific isoforms based on their amino acid sequence, but they also contain isoforms that are synapse specific based on their glycosylation. For example, entactin, found throughout the basal lamina of the muscle is differentially glycosylated (Chiu and KO,1994) in the synaptic basal lamina. It is not known what role this modificationof the entactinplays in the synapticbasal lamina. The mechanism underlying this specific distribution of carbohydrates may be linked to the specific distribution of cytoplasmic enzymes which glycosylate. For example, the enzyme N-acetylgalctosaminyl transferase is localized to the sub-synaptic cytoplasm in muscle (Scott et al., 1990) Analysis of the distribution of synapse specific carbohydrates have been greatly aided by the use of lectins that bind specific sugars. For example the lectins peanut agglutinin (KO1987), Dolichos biflorus agglutinin,and Vicia villosa-34 agglutinin,(WA,Scott et al., 1988) have all been shown to bind to sugars that are concentrated at the neuromuscular junction. The role of these sugars in synaptic structure and function has yet to be determined but it has been found that treatment of myotube cultures with the lectin VVA will inhibit agrin induced aggregation of the postsynaptic apparatus (Martin and Sanes, 1995).
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OTHER SYNAPSE SPECIFIC MOLECULES
Approximately 50 synapse associated molecules have been identified (see introduction; Hall and Sanes, 1993). While it has been shown that these molecules are aggregated at neuromuscularjunctions, it has yet to be determined when and if they are removed following synaptic retraction.
IX. CONCLUSIONS Synapses are highly dynamic structures during development and throughout life. The formation and removal of the synapse is a very precise process, and understanding the cellular and molecular mechanisms controlling the formation and retraction of synaptic structures will be of great importance. Many molecules are specifically concentrated at synapses, and several are modified such that a specialized form of the molecule is synapse specific. Current research has primarily focused on the mechanismsthat control the formation of the synapse; however, the mechanisms that control the breakup of these synapses are poorly understood. It will be interesting to determine whether molecules that are aggregated at different times and rates during synapse formation are dispersed in complementarypatterns. Thus, it may be that the same basic mechanisms that control the formation of the synapse are also involved in the retraction and elimination of that synapse.
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