Many attempts have been made to define early stages and intermediates in amyloid fibrillogenesis that may be susceptible to inhibition. We have developed an in vitro system, based on the use of A beta1-42 peptides, in which the development of prestages of protofilaments and protofilament and fibril formation could, for the first time, be followed by electron microscopy, supported by fluorescence spectrometry. The first recognizable ultrastructures after incubation of A beta1-42 peptides at 37 degrees C were globular subunits (4-5 nm in diameter) that gradually became organized into short protofilaments (30-100 nm), which in turn formed fibrils mainly by lateral association. At this stage, part of the protofilaments were seen first as collaterals protruding from the fibrils and then, as they were gradually incorporated, as buds on the fibril surface. A continuous growth of A beta1-42 fibrils was observed, seemingly originating from a nucleus, which appeared to consist of aggregates of amyloid intermediates. That protofilaments are intermediates also in the in vivo formation of amyloid was supported by the finding that AL fibrils isolated from amyloid tissues also exhibited radiating protofilaments. The demonstrated globular subunits and early formed protofilaments may be attractive targets for inhibition of fibril formation.

Ultrastructural studies of the localization of serum amyloid P component (SAP) in amyloid ®brils have given divergent results. We here report for the ®rst time that electron microscopy of SAP coincubated with Ab 1±42 peptides or with mature Ab 1±42 ®brils, revealed SAP molecules coating the surface of the mature ®brils and that proto®brils of Ab 1±42 did not bind SAP. Also when incubated with extracted amyloid light chain (AL)-®brils the SAP molecules aligned on the ®bril surface. Heparan sulfate proteoglycan bound to the surface of the Ab ®brils with a spacing of about 50 nm. We conclude that SAP does not bind to proto®brils but to the surface of mature Ab ®brils and that it may stabilize and protect the ®brils.

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