Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disease characterized by cognitive decline, and at autopsy, its distinct brain pathologies, neurofibrillary tangles and neuritic plaques. The major protein component of neuritic plaques is a peptide known as amyloid beta (AB), mechanistically linked to AD by virtue of mutations in its precursor protein (APP). These APP mutations can give rise to significant excesses of AB and are causative for early onset familial AD (FAD). The research proposed here involves close collaborations with Dr. C. Finch (USC) and Dr. W. Klein (Northwestern U.). The objective of this research is to define the structure of AB aggregates and precisely determine the molecular interactions responsible for AD-relevant neuronal responses elicited by AB. A further objective is to characterize structural aspects of AB interactions with two other plaque components, apolipoprotein E (apoE) and apoJ, which are elevated in AD brain tissue. Definition of molecular interactions linked to AD-relevant neuronal responses is essential for identification of drug targets to slow or block the progression of the disease pathology. A major impediment to understanding AB's activity is the great difficulty laboratories have in reproducing neurotoxicity or cellular signaling experiments. These difficulties stem from the very nature of AB, which assembles into a variety of distinct forms, only some of which can elicit biological responses. Therefore, another important objective is to ascertain the specific experimental conditions or particular AB peptide analogue structures required to prepare consistently active form(s) of AB.
The specific aims of this research are: 1) Determine the structure of AB fibrils formed under systematically varied conditions, using scanning probe microscopy. Assess structural changes in fibrils of AB analogues containing specific functional changes in AB primary sequence. 2) Define the biologically relevant surface topology of AB aggregates using functionalized AB peptide analogs and chemically or biochemically modified atomic force microscope probe tips. 3) Correlate the neuronal effects of AB (oxidative stress, tau phosphorylation, cell detachment) with fibril structure and topography. 4) Establish experimental parameters and/or prepare AB peptide analogs that enable formation of small, soluble AB aggregates possessing specific, reproducible neurotoxic properties. 5) Analyze the interactions between AB and apoJ or apoE by scanning probe microscopy and use AB analogs to determine the structural requirements for these interactions. Assess biological properties of these complexes. 6) Study AB interactions with neurons by scanning probe microscopy, whole mount electron microscopy and confocal fluorescence imaging. The key methods used in these studies are scanning probe microscopy, and design and synthesis of novel AB analogues. Aggregation kinetics will be measured by absorption/turbidimetry. The biological activities of AB aggregates that will be measured include oxidative stress using the redox substrate MTT, induction of tau phosphorylation in differentiated neuroblastoma cells using a newly developed chemiluminescent sandwich immunoassay, and neuroblastoma disadhesion from fibronectin or laminin. Scanning probe microscopy, whole mount electron microscopy and confocal fluorescence microscopy will be used to study the direct contact and interactions of AB aggregates and neuronal surface molecules.
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