Two new series of monoclonal antibodies (TG and MC) raised against immunoaffinity purified paired helical filament protein (PHF) from Alzheimer's disease (AD) brain are characterized. The antibodies recognize phosphorylated epitopes in PHF and react specifically when M phase cells in culture. Immunoreactivity with the antibodies is markedly increased in cells arrested in mitosis by treatment with nocodazole. These findings led us to examine mitotic activity in human brain. We have found that an antibody MPM-2 which is a marker for a conserved mitotic phospho-epitope displays widespread occurrence of mitotic phospho-epitopes in AD but no reactivity in normal brain. Moreover, we have measured an increase in mitotic kinase activity in AD brain relative to normal. Because of these findings we have hypothesized that mitotic kinases play a role in AD pathology, and that the staining with the TG/MC, and MPM-2 antibodies in AD may be confined to mitotic phospho- proteins. This project focuses on the identification of the antigens recognized by the TG-3 antibody, which is very sensitive and specific for detecting AD pathological changes at the immunocytochemical level and by ELISA. Except for the weak reactivity with the antibody and immunoaffinity purified PHF, TG-3 does not react with any antigen from human autopsy brain. However, it recognizes a 105 kDa protein that appears in cultured human neuroblastoma cells (MSN) exclusively in the M phase of the cell cycle, and detects a similar 105 kDa antigen in human biopsy brain tissue. TG-3 also recognizes an 85 kDa heat-stable protein from cells exposed to the phosphatase inhibitor okadaic acid, and in heat-stable preparations from biopsy brain. It is proposed that the 105 kDa TG-3 antigen is identical to previously described mitotic phosphoproteins of similar molecular weight, and that the heat-stable 85 kDa TG-3 antigen is related to Low Molecular weight MAP-2 (LMW MAP-2). Direct blotting experiments will be conducted to select for those proteins that comigrate perfectly with the TG-3 antigens. The relationship between the comigrating protein and the TG-3 antigen will then be examined using reciprocal immunoprecipitation followed by cross blotting. Antigens tentatively identified in this manner will be further pursued with respect to a more definitive identification and determination of the sequence of the TG-3 epitope. Strategies of peptide mapping and protein sequencing will be employed for identification of the TG-3 reactive proteins and the TG-3 epitope. Molecular approaches will be attempted if an identification is made with respect to previously cloned proteins like LMW MAP-2. The effects of the cell permeable cdc kinase-specific inhibitor, olomoucine, on TG-3 reactivity will be assessed to further establish a functional relationship between mitotic kinases and the TG-3 epitope in intact cells. Studies of TG-3 reactivity in a temperature-sensitive cdc2 mutant mouse cell line will also be undertaken to verify that mitotic kinases phosphorylate the TG-3 epitope. In addition, identified TG-3 antigens will be treated with purified mitotic kinases in order to directly determine whether mitotic kinases produce the TG-3 epitope in these proteins. Identification of the TG-3 antigens and the TG-3 phospho-epitope, and their possible functional association with mitotic kinases will provide a new ground for understanding cellular mechanisms operating in AD.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Research Program Projects (P01)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Albert Einstein College of Medicine
United States
Zip Code
Jicha, G A; Weaver, C; Lane, E et al. (1999) cAMP-dependent protein kinase phosphorylations on tau in Alzheimer's disease. J Neurosci 19:7486-94
Jicha, G A; O'Donnell, A; Weaver, C et al. (1999) Hierarchical phosphorylation of recombinant tau by the paired-helical filament-associated protein kinase is dependent on cyclic AMP-dependent protein kinase. J Neurochem 72:214-24
Dickson, D W (1999) Microglia in Alzheimer's disease and transgenic models. How close the fit? Am J Pathol 154:1627-31
Jicha, G A; Rockwood, J M; Berenfeld, B et al. (1999) Altered conformation of recombinant frontotemporal dementia-17 mutant tau proteins. Neurosci Lett 260:153-6
Kalcheva, N; Lachman, H M; Shafit-Zagardo, B (1999) Survey for CAG repeat polymorphisms in the human MAP-2 gene. Psychiatr Genet 9:43-6
Vincent, I; Zheng, J H; Dickson, D W et al. (1998) Mitotic phosphoepitopes precede paired helical filaments in Alzheimer's disease. Neurobiol Aging 19:287-96
Aronica, E; Dickson, D W; Kress, Y et al. (1998) Non-plaque dystrophic dendrites in Alzheimer hippocampus: a new pathological structure revealed by glutamate receptor immunocytochemistry. Neuroscience 82:979-91
Zhao, M L; Liu, J S; He, D et al. (1998) Inducible nitric oxide synthase expression is selectively induced in astrocytes isolated from adult human brain. Brain Res 813:402-5
Heitner, J; Dickson, D (1997) Diabetics do not have increased Alzheimer-type pathology compared with age-matched control subjects. A retrospective postmortem immunocytochemical and histofluorescent study. Neurology 49:1306-11
Shafit-Zagardo, B; Kalcheva, N; Dickson, D et al. (1997) Distribution and subcellular localization of high-molecular-weight microtubule-associated protein-2 expressing exon 8 in brain and spinal cord. J Neurochem 68:862-73

Showing the most recent 10 out of 71 publications