Investigation on the proteolytic effects of neprilysin and insulin degrading enzyme on the aggregation of Aß40 peptide

Studies have shown that accumulation of beta amyloid peptide (Aβ) has a determining role in the etiology and progression of the Alzheimer s disease. Regulation of the Aß level in brain is a key factor in AD, since its increased production or reduced decomposition leads to the formation of toxic aggregates. The major route of Aß clearance is proteolytic degradation by Aβ degrading enzymes. In this work, we studied the effect of two proteolytic enzymes, neprilysin (NEP) and insulin degrading enzyme (IDE), on the aggregation process and on aggregates of Aβ by three types of approaches: prediction, molecular analysis and toxicity assay. To predict the aggregation behavior of Aβ40 two programs were used; TANGO and AGGRESCAN. Except for a peptide fragment predicted by TANGO to be aggregate prone (resulted from IDE treatment), the two programs generally predicted that cleavage of Aβ40 by each of these enzymes would made Aβ40 to lose its aggregation potential. To study the proteolytic effect of the enzymes in in vitro conditions, first to a 1 μM solution of Aß40, NEP (0.54 nM) or IDE (0.95nM) was added and then incubated for 24 hours at 25°C. Then the effect of NEP and IDE on the already aggregated Aß40 was evaluated. Fluorescence spectroscopy and atomic force microscopy (AFM) were used to detect the aggregates in different experimental setups used in this study. Our findings indicate that aggregating capability of Aß decreases after hydrolysis with NEP and IDE although this reduction was more significant in the case of NEP. The cellular toxicity assay was performed with the use of PC12 cells, either in suspension or in adhered form, and showed that the enzymes could reduce the toxicity of Aβ40 significantly and again NEP was shown to have stronger effect.