The long-term goal of this project is to develop a molecular description of the action of N-cadherin and laminin (LN), representatives of two important classes of proteins that promote axonal growth during development and regeneration. The goals of this proposal are to elucidate the role of Ca2+ signals in neurite growth induced by N-cadherin and LN, clarify the relationship between the Ca2+ signals induced by N-cadherin and those induced by LN, and begin to delineate the role of other intracellular signalling systems in the induction of neurite growth. A strength of the experimental approach is the combination of high resolution single cell imaging, patch clamping, and molecular biology/biochemistry. First, both soluble and bound forms of N-cadherin and LN will be used to effect Ca2+ signals in cultured neurons, and various manipulations and pharmacological reagents will allow the definition of the sources of Ca2+ involved in the induced responses, both in neurons and in non-neuronal cells (where the pathways are expected to differ). Secondly, experimental alterations of the Ca2+ signals, as well as of other second messenger pathways hypothesized to be involved in growth signalling, will be performed for neurons growing on substrates of LN or N-cadherin or neurons contacting artificial LN or N-cadherin """"""""guideposts"""""""". These experiments will allow a determination of the relationship between the messengers and the induction of neurite growth. Finally, cultured neurons and a preparation of isolated embryonic growth canes will each be used to assess the binding interactions of receptors for N-cadherin and LN, and oocytes expressing N-cadherin via injected mRNA will be used to study the function of N-cadherin as its own receptor. The results obtained in this project will provide important information concerning the molecular mechanisms underlying the regulation of axon growth by substrate-bound growth promoting proteins. In addition, they will provide insight into the signaling pathways employed by cadherins and integrins, two classes of cell surface receptor found throughout the developing and adult vertebrate body. A detailed understanding of the normal control of axon growth is necessary in order to understand developmental disorders in which abnormal axon growth is implicated (microcephaly, anencephaly, mental retardation, megacolon, etc.), as well as the regulation of axonal regeneration. Further, advances in our knowledge of intracellular signalling pathways used by cadherins and integrins in neurons is relevant to a broad array of developmental disorders of the nervous system (e.g., spina bifida).
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