TTR and Abeta: An Amyloid Regulatory Network?

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0930102MurphyAlzheimer's disease is the most common age-associated neurodegenerative disease. According to the "amyloid cascade hypothesis", aggregation of a protein, Abeta, is an essential step leading to death of neurons. Deposition of fibrillar aggregates of Abeta in the brain tissue of Alzheimer patients is considered one of the defining characteristic features of the disease. Recent evidence indicates that soluble Abeta oligomers that appear during the course of aggregation, rather than the final mature fibrils, are the most toxic. Studies with transgenic mice indicate that transthyretin, a protein present in blood and cerebrospinal fluid, protects neurons against the devastating damage caused by Abeta aggregates. These studies raise several questions: by what mechanism does transthyretin prevent Abeta toxicity? Is this protective mechanism lost when Alzheimer's disease develops? Are there pharmacological methods to restore transthyretin?s protective abilities?Preliminary data suggest that transthyretin inhibits Abeta toxicity by accelerating Abeta aggregation, thereby reducing the quantity of soluble toxic intermediates. Transthyretin in the blood or cerebrospinal fluid is normally chemically modified at a cysteine residue, and there are over 80 transthyretin mutants that are known to exist. Early data indicate that chemical modification or mutation can strongly influence the interaction of tranthyretin with Abeta, by changing the stability of the transthyretin tetramer. This research will further explore this hypothesis, and will elucidate the mechanism by which transthyretin affects Abeta aggregation. To accomplish this goal, transthyretin will be produced recombinantly and will be chemically modified at the cysteine residue, to mimic the normal blood protein. Additionally, several mutants will be generated by site-directed mutagenesis; these mutants differ by one or two amino acids from the wild-type but have different stabilities. The structure and stability of these transthyretin "variants" will be examined experimentally using mass spectrometry, circular dichroism, fluorescence spectroscopy and related techniques. Aggregation of Abeta in the presence of transthyretin will be measured by laser light scattering, size exclusion chromatography, and electron microscopy. The data will be used to develop mathematical models that describe the association of transthyretin and Abeta and the effect of these associations on Abeta aggregation kinetics. Additionally, small drug-like compounds that bind to transthyretin and increase its stability will be tested to determine if their use enhances the interaction between transthyretin and Abeta. Interestingly, transthyretin itself will aggregate under certain conditions; transthyretin fibril deposition is associated with an age-related disease called senile systemic amyloidosis. Thus, transthyretin is potentially amyloidogenic, yet it protects against the toxicity of another amyloidogenic protein, Abeta. Is it possible that other amyloidogenic proteins also interact with Abeta? Is there an amyloidogenic network of proteins, where proteins regulate each other's aggregation and toxicity? This novel concept will be explored by initiating studies with insulin and related proteins and Abeta. Insulin is a hexameric protein that will dissociate and form fibrils under certain conditions, and there is some limited data suggesting that insulin-related growth factors are, like transthyretin, protective against Abeta toxicity. Factors that regulate the efficacy of transthyretin as a protective agent against Abeta toxicity will be identified. Results from these studies could be used to design compounds with pharmacological activity that stabilize transthyretin and enhance its efficacy. Such compounds would represent a completely novel strategy for preventing Alzheimer's disease. On a more fundamental level, the research could lend support to the notion of a network of amyloidogenic and anti-amyloidogenic proteins that regulate each other's behavior. Such a concept is highly speculative, but identification of such a network, if it exists, would open up many new lines of inquiry and new approaches for preventing a broad range of aggregation-related diseases.Graduate students on this project are broadly educated in modern experimental approaches in protein folding and aggregation, and prepared to take leading roles in academia or in the biotechnology industry. Through active participation in a summer research program, underrepresented minority undergraduates will gain valuable experience and enjoy a sense of accomplishment that will contribute to their continued enthusiasm for research careers.