Inge Grundke-Iqbal, PhD - US grants

The long term objective of this project is to learn the etiology and the pathogenesis of Alzheimer disease and senile dementia of the Alzheimer type (AD/SDAT), a major public health problem in modern society with its rapidly increasing elderly population. The most characteristic lesion in AD/SDAT is the presence of numerous intraneuronal argentophilic fibrillary tangles which are composed of paired helical filaments (PHF). PHF are morphologically unlike any of the normal neurofibers, their origin is as yet not understood. The concentration of Alzheimer neurofibrillary tangles (ANT) strongly correlates to the degree of dementia. Although PHF of the Alzheimer type are not seen in normal young humans or aged animals, ANT/PHF cross-reacting antigens (ANTCA) are present both in normal young human and animal brain. In order to elucidate the role of ANTCA in the pathogenesis of Alzehimer neurofibrillary changes studies will be undertaken to l) biochemically isolate ANTCA polypeptide/s to homogeneity, raise polyclonal antibodies to it, study its localization by immunocytochemistry directly and by absorption of the ANT-staining antibodies with various subcellular fractions, 2)determine its levels by immunoassay in young, adult, aged and Alzheimer brain, and 3) biochemically characterize it in comparison with PHF polypeptides and normal neurofibrous proteins with respect to molecular weight, isoelectric point, amino acid analysis, peptide maps, and end group analysis. Establishment of the identity of ANTCA, its localization in brain and its comparison with PHF polypeptide/s might provide insight as to what determines the formation of ANT/PHF in the affected brain and what is the role of PHF in the pathogenesis of AD/SDAT.

DESCRIPTION (Investigator's Abstract): The long term objective of this proposal is to learn the role of cytoskeletal alterations in the degeneration of neurons in normal aged and Alzheimer disease (AD), a major public health problem in modern society with its rapidly increasing elderly population. AD is characterized clinically by progressive dementia, and histopathologically by an intraneuronal accumulation of masses of paired helical filaments (PHF), disrupting the normal cytoskeleton, and an extracellular deposition of Beta-amyloid in the brain. The presence of PHF correlates with dementia. The PHF are made up of abnormally hyperphosphorylated tau. Hyperphosphorylated tau also occurs in the nonpolymerized form in the neuronal cytoplasm and might be one of the earliest and most critical events contributing to the cytoskeletal pathology in AD. To understand the generation and catabolism of the unpolymerized hypephosphorylated tau (AD P-tau) and its interaction with the normal cytoskeleton, it is proposed to: 1. Determine by radioimmunoassay the relative levels of phosphorylation with site-specific antibodies and compare the agree of phosphorylation at defined sites within AD P-tau and between AD P-tau and PHF tau; 2. Study the interaction of AD P-tau with the microtubule system by determining the interaction kinetics between AD P-tau and normal tau and by analyzing the effect of AD P-tau in vitro on preformed microtubules and in vivo on the microtubule network of cultured cells; 3. Generate hyperphosphorylated tau-in cultured cells by treatment with phosphatase inhibitors and kinase simulators and compare the hyperphosphorylated tau with normal human tau and AD P-tau with respect to its polypeptide pattern and levels of phosphorylation sites, and 4. Study the rate of synthesis and turnover of normal and hyperphosphorylated tau in cultured radiolabeled cells and determine whether tau is degraded mostly by the lysosomal pathway and whether the hyperphosphorylation of tau makes it more resistant to degradation. Phosphorylation sites will be determined by radioimmuno- slot-blot assays. The interaction of AD P-tau with normal tau will be examined in vitro by an overlay radioimmuno assay, and in vivo by intracellular injections and immunocytochemistry. Cell culture models of hyperphosphorylated tau and processing of tau will be investigated by pulse chase studies and Western blots and immunocytochemist.