Thesis (Ph.D.)--Brown University, 2005. / Vita. Thesis advisor: Justin R. Fallon. Includes bibliographical references (leaves 122-130). Also available online.

Recent evidence has challenged the prevalent view that neural factors induce the formation of a de novo postsynaptic apparatus during development of the vertebrate neuromuscular junction. The latest experiments suggest an alternative, muscle-dependent model in which the muscle induces the nascent postsynaptic apparatus and sets the location of the future synapse. Once contacted by the incoming axons, these sites, laid out in a pre-pattern in the central area of developing muscle fibers, mature into synapses by the combined action of neural factors such as agrin and ACh. In this study, I sought to provide a test in mammals for these two models of neuromuscular synaptogenesis. Previously, our laboratory showed that continuous muscle expression of constitutively active ErbB2 (CAErbB2) during embryogenesis leads to synaptic loss, exuberant axonal sprouting and lethality at birth. Here, I transiently induced CAErbB2 during midgestation and examined the process of synapse restoration after inducer withdrawal. Centrallyenriched AChR transcription and AChR clustering were abolished as a result of transient CAErbB2 induction. After inducer withdrawal, synapses were restored but were distributed widely over the entire surface of the diaphragm. Under the nerve-dependent model, this distribution would have been explained by the wide pattern of axonal sprouting triggered by CAErbB2 expression. Yet, in the absence of the nerve, introduced in our transgenic animals by mating to Hb9+/- mice, a very similar, wide distribution of aneural AChR clusters was generated. Thus, even in a case where the central pre-pattern of AChR transcription and clustering is missing, it is the muscle, and not the nerve, that seems to set the site for synapse formation. My results support a muscle-dependent model for the induction of neuromuscular synaptogenesis in mammals.

The neuromuscular junction is a complex structure resulting from the interaction of an innervating neuron and skeletal muscle fiber. The neuron releases a molecule called agrin which acts via a muscle specific tyrosine kinase, MuSK, to initiate the localized differentiation and specialization of the muscle membrane at the synapse. A defining characteristic of the postsynaptic aspect neuromuscular junction is the concentration of acetylcholine receptors (AChRs) at the crests of junctional folds. Also clustered to these sites is the dystrophin associated protein (DAP) complex, a collection of proteins previously associated with muscular dystrophy and synapse formation. A central component of this complex, dystroglycan, had previously been shown to bind to agrin with high affinity. Inhibition studies that blocked agrin binding to dystroglycan indicated that dystroglycan might be required for synapse formation. In addition, several properties of dystroglycan, including its ability to co-cluster with the AChR and to bind both the neural and muscle forms of agrin, made it an obvious candidate as the putative co-receptor for MuSK. Based on these results we have attempted to define the role of dystroglycan in synaptogenesis. / We studied nerve muscle co-cultures derived from Xenopus embryos and found that dystroglycan is present at almost all neural agrin and AChR clusters. This holds true even in developing synapses as well as in extrasynaptic clusters of AChRs. AChR and dystroglycan aggregates can be induced in vitro, by treating myotubes with agrin and/or exogenous laminin. The AChR clusters formed by laminin application were larger and more dense then those formed by agrin treatments and could be inhibited by the addition of anti-laminin antiserum or laminin fragments that do not self-polymerize. In addition, laminin, unlike agrin, was found to induce AChR and dystroglycan clustering independent of MuSK activation. The introduction of an antisense dystroglycan construct into the C2C12 muscle cell line and the resulting reduction in expressed dystroglycan on myotubes also had little effect on phosphorylation but instead reduced the number of agrin induced AChR clusters on myotubes. These finding suggested that dystroglycan is not the co-receptor for MuSK but rather functions at a point in AChR clustering downstream of agrin-MuSK signaling. When similar experiments were conducted on dystroglycan null myotubes we found that these myotubes respond to agrin in a manner similar to wild-type myotubes but that the AChR clusters formed on null myotubes were two to three times larger, half as dense and significantly less stable. Synapses in chimeric mice with dystroglycan deficient muscle were similarly affected. In culture and in vivo the absence of dystroglycan also resulted in the disruption of laminin, perlecan and acetylcholinesterase localization to AChR clusters but did not affect rapsyn or agrin localization. Finally, I present unpublished observations that suggest that the close association of dystroglycan with rapsyn and AChRs may not result from a direct interaction between dystroglycan and rapsyn. From these results we propose that dystroglycan is a key element of a "trap" requir

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 108-114). Also available in electronic version.

Thesis (M.S.)--University of Massachusetts Amherst, 2008. / Includes bibliographical references (p. 29-30).

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / On t.p. "1̳" is subscript. Includes bibliographical references (leaves 114-141). Also available in electronic version. Access restricted to campus users.

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Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 231-257). Also available in electronic version. Access restricted to campus users.

The cytoskeletal component of the muscle membrane, dystrophin and its associated proteins (DAPs), are essential for the maintenance of muscle integrity, since the absence of these molecules results in a variety of muscular dystrophies. The purpose of this work was to create and characterize monoclonal antibodies (mAbs) designed to recognize components of the DAP complex (DAPC), in order to provide tools for the study of its structure and function. / The first mAb generated, 1137, was raised against a 33 amino acid sequence of the core protein at the c-terminus of alpha-dystroglycan (alpha DG), a cell surface member of the DAPC linked to dystrophin via its co-transcript, the transmembrane protein, beta-dystroglycan. 1B7 was used to perform a comparative study in denervated rat muscle tissue in parallel with IIH6, a mAb which recognizes a different, more glycosylated form of alpha DG. The second and third mAbs were raised against a complex of proteins purified by succinylated Wheat Germ Agglutinin (sWGA) following extraction from rabbit skeletal muscle. (Abstract shortened by UMI.)