This project will test the hypothesis that a mitotic posttranslational mechanism is involved in the formation of phospho-epitopes in Alzheimer's disease (AD). A connection between AD and mitosis (M phase) was suggested because antibodies specific for phospho-epitopes found in characteristic neurofibrillary tangles (NFT) of AD brain react exclusively with mitotic cells, and an antibody marker for phospho-epitopes in mitotic cells reacts with NFT in AD, but not with normal brain (Vincent, 1996). As in mitosis, mitotic protein kinase activity isolated with the p13suc1 protein coupled to agarose beads appears to be higher in AD brain than in normal. Although mitotic kinases are not usually present in mature neurons of brain, preliminary studies have suggested that they are activated in NFT and neurons vulnerable to NFT.
In aim 1 of this project, the possibility that a mitotic mechanism is associated with elevated mitotic kinase activity in neurons of AD brain will be explored. The occurrence of mitotic kinases (cdc2, cdk2 and cdk3) and their activating cyclins (A and B1), upstream regulators (the inhibitory wee1 tyrosine kinase and the activating cdc25 phosphatase) and the downstream phosphorylation of nuclear histone H1 will be compared in AD and normal brain. Localization of these proteins to NFT and vulnerable neurons in AD will be examined in fixed brain sections by immunocytochemical and double-labeling techniques. M phase-typical changes in concentration, posttranslational modification, and subcellular localization of the proteins will also be examined immunocytochemically and in biochemical studies using immunoblotting and immunoprecipitation techniques. In addition, activity assays will be done to verify that the mitotic kinases and phosphatase function in AD neurons as they do in M phase.
Aim 2 of this project is to identify the kinase(s) with elevated activity in p13suc 1 fractions from AD brain. Preparative scale isolation of similar preparations will be attempted by affinity chromatography over p13suc1-columns and the kinase with higher activity in these preparations will be identified using a combination of electrophoretic and immunological techniques. These include gel electrophoresis, ELISA, immunoblotting with mitotic kinase antibodies, immunoprecipitation followed by kinase assays and in situ SDS-gel phosphorylation assays. These studies will help determine whether a mitotic posttranslational cascade participates in the accumulation of phosphorylated proteins in neurons of AD brain.
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