Programme Contents

Aggregation state of Aβ peptides in model membranes

Amyloid-beta (Aβ) peptides are postulated to cause loss of nerve cell function in individuals suffering from Alzheimer's disease (AD), where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. However, the molecular mechanism is still unclear and further evidence is required to identify the culprit promoting neuron loss. The peptides aggregate readily and form amyloid fibrils. We have been studying the interaction of Aβ42 peptide with phospholipid membranes using solid-state NMR spectroscopy. However, the aggregation state of the peptide during the NMR experiment is not clear, nor is it obvious that it remains the same throughout the experiment.

The sample preparation is a very crucial aspect in studying the kinetics of Aβ aggregation: use of NaOH, trifluoroacetic acid (TFA) removal, peptide stock solution concentration and temperature are factors promoting completely different aggregation patterns, as observed with either Congo red or thioflavin T fluorescence. When TFA was removed from the peptide, using 5 mM HCl pre-treatment and freeze-drying as a routine procedure, aggregation was not observed, most likely due to the passage from low pH below the PI to a buffered aqueous phase (pH 7.4). Unfortunately, TFA is a strong counter ion that can lead to membrane degradation and dramatic errors in peptide weight estimation. When the TFA was not removed, a monomeric peptide stock solution was prepared in NaOH, and hexafluoroisopropanol was used to dissolve the peptide. Again, little fluorescence was observed from thioflavin T and no difference was observed when experiments were conducted at 28°C or 37°C.

The role of membrane lipids is another factor in Aβ aggregation and kinetic experiments using thioflavin T are being used to reveal if membranes play a critical role in accelerating amyloid formation. Large unilamellar vesicles (LUV) composed of palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylserine (POPS) and cholesterol (Chol) in a molar ratio 1:1:1 were used as a mimic of the neuron cell membranes. A small concentrated volume of peptide in NaOH was externally added to a large buffered solution of LUV in a lipid/peptide molar ratio 30:1 and kept at 37°C with moderate shaking and peptide aggregation was monitored.

Amyloid plaques in the brain are rich in copper, zinc and iron, which bind to Aβ peptides. Aβ and metals have been proposed to promote lipid peroxidation and some evidence suggests that Aβ could drive the metals close to the membrane surface where membrane-oxidizing radicals could be generated. Solid-state NMR is being used to obtain some insights of Aβ-metal interactions with model membranes. Mutation of Tyr 10 replaced by an alanine or oxidation of Met 35, as encountered in AD patients brain, have shown different interactions with POPC/POPS/Chol (1:1:1) at the membrane surface as monitored by changes in the ³¹P chemical shift anisotropy and in the hydrophobic core of the phospholipid bilayers as revealed by the quadrupolar splittings in the deuterium NMR spectra.