Major components of the cytoskeleton; microtubules, neurofilaments and microfilaments play key roles in the structural and functional integrity of nerve cell processes (axons and dendrites). Defects in the assembly and/or interaction of these elements may contribute to the etiology of degenerative neuronal diseases. While substantial information has been accumulated on factors which promote the assembly and stabilization of cytoskeletal components in cell-free systems, the relationship of these factors to the cytoskeleton in differentiating neurons remains elusive. Recently, we reported that zinc is particularly abundant in the shaft of developing nerve processes in areas containing a dense cytoskeletal framework in both transformed and primary neuronal cultures. The association of zinc with developing nerve processes is of particular interest since it has been shown that: 1) zinc is associated with microtubules in a variety of biological systems, 2) zinc promotes the assembly and stabilization of microtubules in cell-free systems, 3) a deficiency of zinc during embryonic development results in brains with developmental anomalies and 4) zinc toxicity results in abnormalities of microtubule structure and leads to the accumulation of neurofilaments in the brains of affected animals. These results suggest that zinc plays a role in the development of nerve cell processes through its interaction with microtubules. The overall objective of this project is to determine the role of zinc in neuronal development. The specific objectives are: 1) to examine the distribution of zinc along with other elements (Ca, Mg, P, S, Cl, K and Na) in nerve processes, 2) to examine the role of zinc in the formation and stabilization of nerve processes and 3) to examine the temporal changes in the uptake, distribution and transport of zinc during neuronal development. To accomplish these objectives, we will employ: a) cultures of transformed and primary neurons as nerve model systems, b) time-lapse cinemicrography to document changes in growth of nerve processes, c) whole-cell transmission electron microscopy and electron-probe microanalysis in tandem to examine the cytoskeletal components and quantitate the levels of zinc and other elements, respectively, and d) radiolabelled zinc to examine the uptake and transport of zinc in developing nerve processes.
