Unraveling an unexpected function of kunitz family members: potent Amyloid-beta aggregation inhibitors

Enhanced metabolism of human amyloid precursor protein (APP) generates truncated Amyloid beta (Aβ) peptide fragments prone to misfold and accumulate as intra- and extraneuronal cell aggregates, among them the Aβ42 neurotoxic specie, which is a hallmark of Alzheimer`s disease (AD).

So far, numerous attempts have been made to target the extracellular Aβ42 aggregates using short peptides and small molecules. Nevertheless, these peptides have a high tendency to self-aggregate which diminishes their inhibitory potency, and small molecules show adverse effects due to nonspecific interactions with other cellular targets. Our new approach therefore includes the development of a non-self-associating agent interacting specifically with Aβ42 and inhibiting its toxicity in neuronal cells.

For that, we have taken advantage of the APP-derived, kunitz-type amyloid precursor protein inhibitor (APPI), to be developed as a protein scaffold with ability to specifically interact with Aβ42 and strongly inhibit the formation of Aβ42 aggregates. Moreover, SH-SY5Y neuroblastoma cell line treated with both extracellular APPI and Aβ42 exhibited increased cell viability relative to cells treated with pre-formed Aβ42 aggregates. Using confocal microscopy, we observed that extracellular Aβ42 permeated and accumulated as insoluble inclusion bodies within the cells, whereas treatment with APPI abolished the intracellular accumulation of Aβ42, which explains the protective effect APPI has against neurodegeneration in these cells. A Thioflavin T (ThT) assay, which confirmed that APPI reduced Aβ42 aggregation in vitro, provided the mechanistic explanation for the effects APPI has in cells. Taken together, these results provide a strong experimental evidence that APPI is a promising candidate for specific inhibition of Aβ42 aggregation and as a potent AD therapeutic.