Development of the mammalian brain proceeds via a series of carefully regulated steps; interference with any of these steps has profound consequences on normal development. Critical to normal morphogenesis is precise temporal expression of cell adhesion molecules (CAMs) and of cytoskeletal modifications which are hallmarks of these steps. Data obtained during the previous funding period indicate that while microtubules are susceptible to methylmercury (MeHg), CAMs are also critically vulnerable to MeHg at specific developmental stages. There is also clear evidence that targeting of CAMs by MeHg leads to an interruption of the essential functional linkage between cell adhesion molecules and the cytoskeleton. Since the nature of impaired brain morphogenesis reflects the developmental step disrupted, and CAMs are differentially expressed at the various stages, it is hypothesized that disturbance of brain development by MeHg is caused by perturbation of CAMs at critical stages or """"""""windows"""""""" of neural morphogenesis. The relationships between MeHg, CAMs, and CAM-cytoskeletal linkages will be investigated by addressing the following specific aims: 1) To determine if MeHg acts directly on CAMs, or via CAM biosynthesis, to differentIally alter the expression and function of CAMs at particular developmental stages. 2) To ascertain the mechanism by which MeHg disturbs the linkage between CAMs and cytoskeleton. 3) To develop a model which permits experimental control of CAM expression (using cDNA transfection techniques) to examine critical susceptibilities of CAMs to MeHg and subsequent influences on cytoskeleton. This project represents the first systematic study of the effects of a toxic metal on CAMs during brain development.
The specific aims will be addressed using a combination of contemporary molecular and cellular techniques applied to models at the cell (cell culture), tissue (hippocampal or cerebellar slice), and whole animal levels of organization. Members of the calcium-dependent (N-cadherin) and calcium- independent (NCAM, L1) families of CAMs will be examined to determine the effects of MeHg on their expression and modulation, and the integrity of their linkage to cytoskeleton. Immunofluorescence and immunohistochemical microscopy, in situ hybridization, immunoblot analysis, quantitative ELISA, and functional binding assays will be used to provide overlapping approaches to unravelling MeHg-CAM interactions. Finally, the relationship between CAMs and cytoskeleton under normal and toxicant- conditions will be determined in cultured neurons and in 3T3 fibroblasts transfected to express specific CAMs prior to and following chemical perturbation of specific cytoskeletal elements.
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