The long-term objectives of this research are to elucidate the cellular and molecular mechanisms of development, maintenance and plasticity of the synapse. The present proposal will focus on the involvement of extracellular matrix (ECM) glycoconjugates in these processes at the neuromuscular junction (NMJ) using peanut agglutinin (PNA) as a molecular probe.
The specific aims are: (I). To test the hypothesis that expression of synaptic ECM precedes the nerve terminal outgrowth during synaptic remodeling. (A) Identified NMJs will be observed repeatedly in vivo with video-enhanced microscopy followed by electron microscopy. In addition, whether Schwann cell processes lead the nerve terminal will be studied. (B) The dynamic relationship among nerve terminals, synaptic ECM and acetylcholine receptors during the active growth of sprouting will be examined in vivo. (II). To characterize PNA-binding molecules (PNA-BMs) and test their functions. (A) Affinity chromatography, gel electrophoresis and lectin blotting will be used to isolate and identify the PNA-BMs. (B) Antibodies against PNA-BMs will be produced and immunocytochemistry and immunoblotting performed. (C) The effects of affinity-purified PNA-BMs and functional perturbation with antibodies on neurite outgrowth in vitro will be examined. (D) Antibodies will be applied to perturb the function of PNA-BMs in synapse formation and maintenance in vitro and in vivo. The proposed research would provide novel concepts on the role of the ECM and Schwann cells during synaptic remodeling in living adult animals. The proposal would also characterize new synapse-specific ECM molecules and test the function in formation, plasticity and maintenance of the NMJ. The result may lead to a better understanding of neuromuscular diseases and restoration after trauma. The study may also provide new thinking on learning and memory, which may involve remodeling of synaptic connections in the brain.
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