Aggregation of Aß42 is strongly influenced by Gly33 of the GxxxG motif and an inhibitor peptide which mimics Gly33 substitutions

Abstract

Previously, we have studied the influence of the Aß GxxxG motif on the oligomerization and toxicity of Aß42 peptides. Synthetic Aß42 peptides with substitutions of G33 to an Ala or Ile easily aggregated into higher oligomeric forms compared to wt peptides. Particularly, we identified Aß42wt tetramers as the most toxic form whereas G33A or G33I peptides were not toxic independent of the oligomeric state. In the present study we performed detailed studies to investigate structural requirements of Aß42wt toxicity. A small peptidic inhibitor molecule was used as a valuable tool to study oligomerization and fibril formation of Aß42. In addition we found an effect of the inhibitor on the toxicity of peptides. Size exclusion chromatography (SEC), electron microscopy (EM) and atomic force microscopy (AFM) were used to analyze the oligomerization of low-n forms, proto fibril- and fibril formation. Primary neurons were incubated with freshly dissolved or aggregated Aß42 in the presence or absence of the inhibitor peptide to determine neuronal viability. Whereas Aß42wt peptides formed mature fibrils substitutions for G33 arrested the process of aggregation in a certain stage of proto fibril formation as observed by EM and AFM. G33 substitution peptides formed protofibrils with a similar diameter as the wt peptide but their lengths were drastically reduced. SEC of freshly dissolved substitution peptides revealed the presence of mainly higher oligomeric forms compared to the wt peptides. However, oligomeric forms of Aß42wt peptides could be stabilized when co-incubated with an inhibitor. Those aggregates structurally resembled G33 substitution peptides when analyzed by EM and AFM. Most interestingly, the co-incubation of Aß42wt with the inhibitor neutralized the toxicity of low-n oligomeric forms. We hypothesize that a direct interaction of the inhibitor with the GxxxG motif interferes with the toxicity at the cellular level. Our data indicates that Aß aggregation is regulated by the GxxxG motif, either when G33 is substituted or small molecules may directly bind to the C-terminal region of Aß42. The inhibitor is a promising tool to antagonize toxicity of low-n oligomeric forms of Aß42wt to living cells.