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

Sammanfattning: Alzheimer's disease is a progressive neurodegenerative disease, mostly affectingelderly. The invariable deposition of protease-resistant fibrils of Alzheimer amyloidß-peptide (Aß) in the parenchyma and blood vessels of the brain is a centralevent. The aim of this study was to investigate whether Aß develops proteaseresistance upon polymerization and whether Aß may be generated through non specificproteolysis of a polymerized precursor, to identify Aß-Aß binding and fibrilforming 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 polymerizedprecursor. A C-terminal fragment of APP was purified and allowed to polymerize. Fibrilsremained after digestion with proteinase K, an enzyme capable of cleaving most peptidebonds. The fibrils were dissolved and subjected to gel electrophoresis, showing anAß immunoreactive band comigrating with synthetic Aß. A sequence critical for Aß-Aß binding was identified by incubating decapeptidescorresponding to various Aß sequences with labeled Aß By systematicallytruncating binding peptides, the shortest sequence mediating intact Aß bindingwas found to be Aß16-2o Short peptides containing this sequence inhibited fibrilformation. Further studies showed that Aß16-2o bound to the homologous regionin Aß. The Aß16-2O sequence was used as a labeled probe to search for bindersin a combinatorial library consisting of pentapeptides composed of D-amino acids.The most efficient binders were incubated with Aß and found capable of inhibitingfibril formation. Hence, short Aß16-2o containing peptides can be used to identifyinhibitors of Aß fibril formation in combinatorial libraries. Fluorescence correlation spectroscopy (FCS) was used to follow the polymerizationof Aß in solution. Initially, only monomers/dimers were observed and polymerizationwas 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 formedaggregates were partly dissolved after addition of an inhibitor. Labeled inhibitorbound to monomeric/dimeric Aß as well as aggregates and was displaced by unlabeledinhibitor. The shortest fibril forming sequence containing the Aß-Aß binding motifwas identified by systematically elongating and truncating sequences containing Aß16-2o.The shortest sequence forming Aß-like fibrils was Aß14-23. Substitutionsand truncations in this peptide yielded ultrastructurally different aggregates, noneresembling Aß fibrils. A model with favorable hydrophobic and ionic interactionsbetween two Aß14-23 peptides in an antiparalell ß-sheet configurationwere 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 theseinteractions are present also in Aß fibrils. Key words: Alzheimer's disease, amyloid, fibrils, inhibitor, protease resistance,electron microscopy, fluorescence correlation spectroscopy. ISBN-9 1 -628-2824-X