CORE--BRAIN MICRODISSECTION, RNA AND PROTEIN ANALYSIS
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Specific cytoarchitectural areas, subfields, and even lamina develop Alzheimer pathological changes, while other are spared. A pattern of hierarchical vulnerability defines areas that are at risk. This proposal will test hypotheses about molecular characteristics that could underlie this pattern of vulnerability. Our previous studies show neurons that develop neurofibrillary tangles (NFT) occur mainly in specific lamina in limbic and association cortices. Senile plaques (SP) are found in a different but consistent hierarchical pattern, with some brain areas substantially more affected than others. We now propose to combine quantitative anatomic analyses of pathological changes, immunohistochemical and in situ hybridization studies, and RNA analyses from microdissected brain regions to examine how neurons that develop NFT differ from those that do not, and how regions that accumulate SP differ from those that are spared. In the first aim, we will pursue preliminary results which that a change occurs in AD in the pattern of tau isoforms expressed. The second and third aims focus on possible tau kinases. Recent studies show that the MAP Kinase (MAPK) family of Pro directed Ser/Thr kinases (ERKs) phosphorylate tau in vitro in a pattern resembling the changes that occur in NFT. We will study ERKs 1 and 2, ERK3 and the p54 MAPKs. We will test the hypothesis that p54 MAPK phosphorylates tau in a PHF tau fashion. Glycogen synthase kinase 3 has also been implicated in tau phosphorylation. These kinases are all highly expressed in brain, but the neuroanatomical regional and cellular distribution of their expression is unknown. We will study the normal patterns of their expression a swell as determine whether or not changes occur in neurons that develop NFT and in the microenvironment surrounding SP. Pilot experiments will be aimed at determining the activation status of these kinases in the AD brain. In AIM 4, we continue our studies of molecules potentially related to SP formation. Our previous studies, in collaboration with Dr. Tanzi (project 4), analyzed expression of APP major splice forms and found no difference between AD and controls. We now turn to the anatomic distribution and expression of a novel isoform of APP, L-APP (lacking exon 15), as well as new members of the APP family, APLP1 and APLP2. We have observed that nitric oxide synthase neurons are spared in AD. In Aim 5, in collaboration with Dr. Young, (project 1), we will determine the phenotype of these spared neurons in terms of tau, kinase, and glutamate receptor expression. In sum, the overall goal of this project will be to advance the profile of vulnerability from anatomical description towards molecular phenotype by generating profiles of tau, the newly described ERK family of protein kinases, and APP family members in vulnerable and spared areas and cells.
This core will provide a central resource for reagent synthesis, tissue preparation, and quantitative RNA and protein analysis for all 5 projects. DNA oligonucleotides will be synthesized using an Applied Biosciences 392 DNA synthesizer and purified by gel electrophoresis. These will be used for in situ hybridization protocols in projects 1,2, and 4. RNA and protein will be isolated from tissue samples used in each of the projects (human brain, experimental animals, or tissue culture material). Brains will be microdissected to allow analysis of discrete anatomical regions. RNA and protein separation will be carried out by RNAse protection assays and Western blots. Quantification will be by autoradiography and densitometry using an LKB Ultrascan Laser Densitometer. Providing these quantitative assays as a core service will enhance each project, and be cost effective compared to duplicate analytical setups in each laboratory. Particularly with regard to the human material, the core will allow for optimal use of scarce resources. Furthermore, in the course of these studies, the core will generate RNA and protein samples from carefully microdissected brain regions of clinically well characterized patients and young and old experimental animals. This will be an invaluable resource and will be of great use as these studies of aging and Alzheimer disease evolve.
