Molecular basis and pharmacological implications of Alzheimer amyloid ß-peptide fibril formation

Alzheimer's disease is a progressive neurodegenerative disease, mostly affecting elderly. The invariable deposition of protease-resistant fibrils of Alzheimer amyloid ß-peptide (Aß) in the parenchyma and blood vessels of the brain is a central event. The aim of this study was to investigate whether Aß develops protease resistance upon polymerization and whether Aß may be generated through non-specific proteolysis of a polymerized precursor, to identify Aß-Aß binding and fibril forming sequences in Aß and define the molecular basis of Aß polymerization, and to find inhibitors of fibril formation.

To investigate if Aß fibril formation is sufficient to acquire protease resistance, non-polymerized and polymerized Aß were treated with an array of proteases. Non-polymerized Aß was degraded whereas fibrils were resistant to proteolysis. The proteases generating Aß from its precursor, APP, have not been identified. We show that Aß can be generated through nonspecific proteolysis of a polymerized precursor. A C-terminal fragment of APP was purified and allowed to polymerize. Fibrils remained after digestion with proteinase K, an enzyme capable of cleaving most peptide bonds. The fibrils were dissolved and subjected to gel electrophoresis, showing an Aß immunoreactive band comigrating with synthetic Aß.

A sequence critical for Aß-Aß binding was identified by incubating decapeptides corresponding to various Aß sequences with labeled Aß by systematically truncating binding peptides, the shortest sequence mediating intact Aß binding was found to be Aß 16-20 Short peptides containing this sequence inhibited fibril formation. Further studies showed that Aß 16-20 bound to the homologous region in Aß. The Aß 16-20 sequence was used as a labeled probe to search for binders in a combinatorial library consisting of pentapeptides composed of D-amino acids. The most efficient binders were incubated with Aß and found capable of inhibiting fibril formation. Hence, short Aß 16-20 containing peptides can be used to identify inhibitors of Aß fibril formation in combinatorial libraries.

Fluorescence correlation spectroscopy (FCS) was used to follow the polymerization of Aß in solution. Initially, only monomers/dimers were observed and polymerization was found to proceed over large aggregates, via small aggregates into mature fibrils. The amounts of aggregates were reduced in the presence of an inhibitor and formed aggregates were partly dissolved after addition of an inhibitor. Labeled inhibitor bound to monomeric/dimeric Aß as well as aggregates and was displaced by unlabeled inhibitor.

The shortest fibril forming sequence containing the Aß-Aß binding motif was identified by systematically elongating and truncating sequences containing Aß 16-20. The shortest sequence forming Aß-like fibrils was Aß 14-23. Substitutions and truncations in this peptide yielded ultrastructurally different aggregates, none resembling Aß fibrils. A model with favorable hydrophobic and ionic interactions between two Aß 14-23 peptides in an antiparalell ß-sheet configuration were subjected to molecular modeling. Two dimers were aligned to form a tetramer, which could be the smallest repeating unit in fibrils. It is suggested that these interactions are present also in Aß fibrils.