Localization of the APC tumor suppressor protein in neurons and identification of a novel oligodendrocyte marker.

摘要

This thesis represents two lines of work. The majority of this work represents studies conducted on the adenomatous polyposis (APC) protein in the brain. In the second, unrelated portion, I report a previously completed study assessing the regulation of a novel immediate early gene, arc, by cocaine.; Identification of mutations in the APC tumor suppressor gene in a substantial percentage of colon cancers has stimulated intense interest in elucidating the role of APC in intestinal epithelial cells. As the brain expresses extraordinarily high levels of APC mRNA when compared to peripheral tissues, including the colon, we wondered what role this protein may play in the nervous system. Because localization of proteins in the brain often provides important clues to their function, we began by conducting immunohistochemical studies in rat brain.; Initial studies using a panel of commonly-used N-terminal antibodies revealed intense labeling of glia, most notably oligodendrocytes. The antibodies used recognize distinct epitopes, and have also been shown to detect APC in transfected cells. However, since our initial in situ hybridization studies revealed high levels of APC mRNA in neuronal populations, we sought to resolve this discrepancy between protein and mRNA localization. By using a variety of approaches, we suggest that the glial immunostaining observed with N-terminal antibodies reflects interaction with a cross-reacting antigen(s), rather than localization of APC. As there is a great need for oligodendrocyte markers that stain the cell body while leaving the myelin unstained, we proceeded to characterize this labeling, which we call CC1 to reflect the antibody which yielded the most intense staining. Immunocytochemistry combined with in vivo bromodeoxyuridine labeling revealed that CC1 staining only appears after the oligodendrocyte precursors have exited the cell cycle.; To define the localization of APC protein in brain, we undertook immunohistochemical studies using C-terminal antibodies. These studies revealed labeling of selected neuronal populations, in a pattern that matches that detected by the in situ hybridization studies. Particularly intense labeling was observed by projection neurons in several brain areas, including Purkinje cells, mitral cells of the olfactory bulb, hippocampal pyramidal neurons and pyramidal neurons located in layer V of the neocortex. In addition to labeling of the cytoplasm, APC staining extended into the dendritic and axonal processes. In addition, preliminary studies using immunoblot analysis of post-synaptic density fractions demonstrated that APC is associated with these organelles. Previous biochemical studies have shown that APC binds to {dollar}beta{dollar}- and {dollar}gamma{dollar}-catenin, intracellular components of the cadherin-based cell-cell adhesion system, as well as hDLG, a PDZ protein implicated in proper synapse formation and function. Those findings and our results indicate that APC may play an important role in regulating neuronal structure and function.