REGULATION OF MAP2 IN BRAIN

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Microtubule Associated protein 2 (MAP2), is a complex protein, which co-purifies with microtubules of the neuronal cytoskeleton, and seems to have an important role in the organization of microtubules and their interactions with other proteins and organelles. MAP2 is of particular interest in brain because of its abundance, its specific localization in dendrites, and the regulation of its expression during brain development and neuronal differentiation in culture. The molecular mechanism of MAP2 expression in brain and in PC12 cells, will be studied at the level of protein systhesis, mRNA and the genes that code for this protein. For that purpose, MAP2 cDNA will be isolated, by screening lambda gt11 brain libraries with highly specific polyclonal and monoclonal antibodies. The clone identity will be confirmed by hybrid selection, and by probing RNA derived from different tissues and cells lines with the cDNA. Initial observations showed a significant induction of MAP2 levels during treatment of PC12 cells with NGF. The mechanism of this induction will be analyzed by the temporal correlation between the rate of MAP2 synthesis (by pulse labeling), the translational activity of MAP2 mRNA (by in vitro translation), and the steady state levels of the mRNA (by Northern blots). Similar techniques will be used to follow the developmental regulation of MAP2 isotypes (MAP2a and MAP2b) in rat brain; specifically, whether these different isotypes are the product of precursor processing, or translation of two distinct transcripts. The genetic complexity of MAP2 will be examined by Southern blots, to determine whether the MAP2 gene is part of a family that shares structural and functional domains. Finally, the appearance and localization of MAP2 mRNA, measured by in situ hybridization, will be compared to the immunocytochemical distribution of MAP2 in dendrites, thus, testing the hypothesis that polysomes located near dendritic spines may be engaged in local protein synthesis. The objective of this research is to understand the regulation of cytoskeletal proteins during neuronal differentiation, and the role they play in the morphological abnormalities associated with many neurological disorders, particularly, in relation to dendrite dystrophy associated with mental retardation.