Synthetic betaAP 1-42 spontaneously aggregates into fibrillar structures which resemble those found in amyloid plaques and cerebrovascular amyloid deposits Of Alzheimer's diseased brains. In contrast to unstructured monomeric betaAP, fibrillar aggregates Of betaAP 1-42 are toxic to neurons suggesting that the neurotoxicity Of betaAP depends upon its state of aggregation. At the nM concentrations of betaAP found in both AD and healthy control patients' cerebrospinal fluid, we have found that betaAP aggregates very slowly. Consistent with a nucleation-dependent mechanism, we observe that addition of small amounts of preformed betaAP aggregates (that is """"""""betaAP-seed"""""""" material) nucleates the rapid accretion of additional betaAP molecules into a growing """"""""one-dimensional crystal."""""""" If we could stabilize this betaAP seed material, then we could readily study the functional and structural properties of seeds. Glucose reacts with primary amino-groups to form Advanced Glycation Endproducts or AGEs which can cross-link betaAP through AGE"""""""" bridges and stabilize its conformation. When pre-formed aggregates of AGE-modified betaAP or """"""""AGE-betaAP seeds"""""""" are added, they nucleate more aggregation of monomeric betaAP than unmodified """"""""betaAP seeds"""""""" or no seeds. We have also prepared amyloid plaque-enriched fractions from AD brain and from age-matched healthy brains and measured their protein content and their AGE content. We find that plaque-enriched fractions from AD brain samples contains more AGEs per mg protein than plaque-enriched fractions prepared from age-matched, healthy brains. Based on these findings, we propose to study the functional, structural and conformational characteristics of """"""""seeds"""""""" which accelerate the aggregation of unstructured betaAP into fibrillar beta-amyloid and then measure the in vitro and in vivo neurotoxicity of this beta-amyloid. Since AGE- modification enhances the potency with which seeds promote betaAP aggregation, is known to stabilize long-lived proteins, and is found in plaque fractions, we also propose to measure AGE-betaAP levels in the cerebrospinal fluids from patients with AD, patients with non-AD dementias and from age-matched, healthy patients.
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