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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG012721-04
Application #
6055396
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1996-09-30
Project End
2001-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Washington
Department
Pathology
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Park, Kevin H J; Lu, Ge; Fan, Jing et al. (2012) Decreasing Levels of the cdk5 Activators, p25 and p35, Reduces Excitotoxicity in Striatal Neurons. J Huntingtons Dis 1:89-96
Park, Kevin H J; Vincent, Inez (2008) Presymptomatic biochemical changes in hindlimb muscle of G93A human Cu/Zn superoxide dismutase 1 transgenic mouse model of amyotrophic lateral sclerosis. Biochim Biophys Acta 1782:462-8
Zhang, Min; Hallows, Janice L; Wang, Xuezhen et al. (2008) Mitogen-activated protein kinase activity may not be necessary for the neuropathology of Niemann-Pick type C mice. J Neurochem 107:814-22
Park, Kevin H J; Hallows, Janice L; Chakrabarty, Paramita et al. (2007) Conditional neuronal simian virus 40 T antigen expression induces Alzheimer-like tau and amyloid pathology in mice. J Neurosci 27:2969-78
Hallows, Janice L; Iosif, Robert E; Biasell, Rebecca D et al. (2006) p35/p25 is not essential for tau and cytoskeletal pathology or neuronal loss in Niemann-Pick type C disease. J Neurosci 26:2738-44
Rashidian, Juliet; Iyirhiaro, Grace; Aleyasin, Hossein et al. (2005) Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo. Proc Natl Acad Sci U S A 102:14080-5
Zhang, Min; Li, Jin; Chakrabarty, Paramita et al. (2004) Cyclin-dependent kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal lesion formation, and motor defects in Niemann-Pick Type C mice. Am J Pathol 165:843-53
Hallows, Janice L; Chen, Ken; DePinho, Ronald A et al. (2003) Decreased cyclin-dependent kinase 5 (cdk5) activity is accompanied by redistribution of cdk5 and cytoskeletal proteins and increased cytoskeletal protein phosphorylation in p35 null mice. J Neurosci 23:10633-44
Lu, Kun Ping; Liou, Yih-Cherng; Vincent, Inez (2003) Proline-directed phosphorylation and isomerization in mitotic regulation and in Alzheimer's Disease. Bioessays 25:174-81
Vincent, Inez; Pae, Chong In; Hallows, Janice L (2003) The cell cycle and human neurodegenerative disease. Prog Cell Cycle Res 5:31-41

Showing the most recent 10 out of 17 publications