Investigation of Cytotoxicity and Ion Flux Induced by Various Aggregation States of Amyloid-Beta Peptides.

Abstract

The pathological feature of Alzheimer’s disease (AD) involves accumulation of amyloid-beta (Ab) peptides into plaques in brain tissues. Two species of Ab peptides, Ab1-40 and Ab1-42, have been identified as the major components of the amyloid plaques. Most studies have reported that the intermediate oligomeric forms of Ab are responsible for neurodegeneration; the exact mechanisms of neurotoxicity, however, are still unclear. An increasing number of evidence indicates that Ab peptides can form pores in neuronal membranes and cause uncontrolled ion flux through cellular membranes, leading to a disruption of the ion homeostasis. In this thesis, with regard to a controversy on the mechanism of Ab-induced ion flux: ion channel formation versus thinning membrane hypotheses, we provided an evidence that Ab can form pores which permit ion flux through the artificial lipid and neuronal membranes, while the proposed membrane thinning effect was due to the residual amounts of the solvent hexafluoroisopropanol, which was used in the preparation procedure. In addition, we used planar lipid bilayer recordings to study the formation of ion channel, and cytotoxicity assays to determine the toxicity of Ab from various preparation methods of Ab. We characterized the aggregation states of Ab using biochemical and biophysical techniques to correlate the relative abundance of aggregated Ab species with pore formation and cytotoxicity. Our statistical analyses revealed that pore formation was correlated most strongly with tetramers to hexamers, while cytotoxicity correlated with tetramers to 18 mers. The partial overlap of Ab oligomers that induced the highest probability of pore formation with those that were most toxic suggests that pore formation is likely a contributing mechanism to the toxicity of Ab. In the second part of this research, we characterized two synthetic molecules containing oligo(ethylene glycol), which self-assemble to form ion channels across lipid membranes. We found that these molecules also exhibited antibacterial activity against gram-positive bacteria, which might be appealing as a starting material for the development of antibiotics.