PHOTO-INDUCED DAMAGE OF AMYLOID ß BY THE CHLORIN e6 PHOTOSENSITIZER

Inhibition of amyloid b (Aβ) aggregation and destabilization of toxic Aβ oligomers are promising approaches for the prevention and therapy of Alzheimer`s disease (AD). Compounds that block Aβ aggregation have been identified in many studies, for example, peptides containing a recognition element KLVFF (residues16-20 in Aβ) were developed to bind Aβ and disrupt its aggregation, thus protecting neurons from Aβ-associated toxicity. Herein, we utilize the high affinity of the hemin-like porphyrin, Chlorin e6 (Ce6), to Ab and its well-known photosensitizing property to selectively damage Ab upon illumination with visible light. The Thioflavin-T (ThT) assay as well as the transmission electron microscopy (TEM) studies demonstrated that Ce6 exhibits potent antiamyloidogenic activity in dark, however, brief illumination of the Ab-Ce6 mixture dramatically increases its activity. Ce6 also protected rat pheochromocytoma PC-12 cells from Ab toxicity in dark, however illumination with visible light significantly increased this activity.

Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying Ce6 antiamyloidogenic activity. NMR spectroscopy indicated that Ce6 selectively binds the His residues in positions 13 and 14 of Ab. A significant loss of His residues was observed by NMR as well as amino acid analysis upon irradiation of the Ab-Ce6 mixture with light. Overall, the NMR and the amino acid analysis together with the PICUP results demonstrated that irradiation of Ce6 in the presence of Aβ induces intra-molecular cross-links and also modifies His residues in Aβ sequence, which can be responsible for its photo-induced antiamyloidogenic activity.