application) The morphology of an amyloid fibril has traditionally been analyzed by electron microscopy. However, this method requires extensive sample preparation and staining with, for example, uranyl acetate, which may affect morphology. Atomic force microscopy (AFM) allows sample analysis under conditions which may closely relevant physiological conditions. In addition, AFM allows the elucidation of the pathway of amyloidogenesis, since images can be obtained rapidly, without interrupting the process. Most current approaches to elucidation of AD amyloidogenesis assume that a given variant of the A beta protein forms a single fibrillar species. However, considerable evidence suggests that amyloid fibril morphology in Alzheimer's disease is subtly varied. These variations may have important biological consequence, affecting, for example, their interactions with neuronal surfaces. Therefore, it is important to elucidate these morphological differences and to determine the kinetic details of the protein aggregation pathways which lead to each morphology. AFM is perfectly suited for this task since, unlike electron microscopy, sample preparation is minimal and experiments can be conducted under physiologically-relevant conditions. The applicants propose to utilize AFM (1) to determine the structural morphology of amyloid fibers obtained from different precursors and pathways under physiologically relevant conditions, (2) to elucidate the in vitro growth kinetics of amyloid and to assess how endogenous brain proteins and small molecule drug candidates obtained from industrial collaborators affect these kinetics, (3) to probe directly the binding energetics of small molecules to amyloid fibers, and (4) to characterize the amyloid fibers and diffuse amyloid from post-mortem brain tissue. The results of these studies will provide a detailed understanding of the mechanism of in vitro amyloid formation at the nanometer length scale, and furthermore, will assess the effects and origin of small molecule drug candidates in inhibiting amyloidogenesis and the relevance of in vitro data to amyloid formed in vivo.
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