Cytotoxicity experiments were performed with both the forms of the peptides at different stages of their aggregation. While redADan does not undergo disulfide bond formation spontaneously 1 mM DTT which by itself is not cytotoxic to neuronal
E7080 was included in the cytotoxicity assay to forestall even the chance formation of a disulfide bond in redADan. It is observed that the fresh peptide in both the forms (soluble and non-aggregated) is more toxic compared to their completely aggregated counterparts. In fact the small spherical protofibrillar masses are also more toxic compared to the completely aggregated fibers. Comparing the oxidized and the reduced peptides we note that the redADan peptide is more toxic than its oxidized countepart (oxADan) at all stages of aggregation. Thus, for the fresh peptides the lethal activity for the redADan starts as soon as the peptide is added to the culture and lethality to the extent of 40% is observed at the end of three hours with marked change in the morphology of the
cells (Fig. 4A). For the oxidized form the viability of the cells is almost similar to the untreated control cells at 3 h. Moreover the cells also retain their morphology (Fig. S3a). However, after 6 h of incubation with oxADan, mild changes in the cellular morphology occur along with death of some cells. Overall the viability of the cells treated with redADan and oxADan after 6 h is 42% and 67%, respectively. The fresh redADan shows 30% cell viability after 24 h of incubation, while oxADan shows 60% cell viability under identical conditions (Fig. 4A).
