Designed Cell-Penetrating Peptides for Inhibition of Amyloid-Beta (Aβ) Amyloid Formation and Cytotoxicity

Several neurodegenerative diseases are characterized by self-assembly of proteins or peptides into aggregates termed amyloid, which share various physicochemical features: a fibrillar morphology, a predominantly β-sheet secondary structure, insolubility in common solvents and detergents, and protease resistance. These so-called amyloid diseases include Alzheimer's disease (AD), Huntington's disease, Parkinson's disease and prion diseases. Despite intense research efforts over several decades, the pathophysiology and interlinkage of these diseases remains unresolved. This is mainly due to the transient nature of the different aggregation states along the amyloid formation pathway, which impedes identification of the cytotoxic species. Recently, a peptide derived from the unprocessed N-terminus of cellular prion protein (PrPC) was shown to function as a cell-penetrating peptide (CPP) and to significantly reduce levels of the disease-associated scrapie isoform (PrPSc) in prion-infected cells. This peptide comprises two segments: a hydrophobic signal sequence, to which the CPP property is attributed, followed by a polycationic nuclear localization signal (NLS)-like sequence that interacts with PrPSc. Interestingly, the anti-prion efficiency of the peptide could be enhanced by substitution of the PrPC signal sequence with a more flexible signal sequence from the neural cell adhesion molecule-1 (NCAM-1). Here, we extended the study towards amyloid-beta (Aβ) aggregation, which is associated with AD. We investigated the amyloid formation and cytotoxicity of the Aβ-inhibitor complexes using thioflavin T fluorescence assays, AFM, NMR, cell proliferation assays and fluorescence imaging methods. We report that the NCAM-1-based inhibitor stabilizes Aβ in a non-amyloid state and protects neuronal cells against Aβ-induced neurotoxicity. Moreover, we show that the replacement of the PrPC NLS-like sequence with a NLS-like segment from Aβ results in similar CPP-functionality and anti-Aβ effects. Our findings reveal a general underlying principle for the inhibition of pathogenic protein aggregation that will allow for the design of a ‘universal’ peptide-based inhibitor.