Chirality, Alzheimer’s Disease, & Amyloid Beta

Chirality is of para­mount im­por­tance to all living systems. It is a property of all principal bio­mo­le­cular buil­ding blocks, i.e., amino acids except glycine, sugars and nucleosides, as well as lipids. Continuous improvement of preparative metho­dologies meanwhile allows increasingly complex mole­cules to be synthesized, which includes so­phi­s­ti­ca­ted mirror-image biomolecules, such as proteins.

Aggregation-prone (amyloidogenic) polypeptides are produced by living systems, often as cleavage products of substantially larger protein precursors. Whereas their functions in health are challenging to study and not always well understood, it is widely accepted that an imbalance between their production and clearance can trigger a range of pathological conditions, including Alzheimer’s Disease (AD / amyloid β, Aβ), Huntington’s Disease (HD / the huntingtin protein) and Type 2 Diabetes (T2D / amylin). A feature that is common to all those peptides is the high polydispersity across both aggregate size and shape, with distinctions frequently made between oligomers, protofibrils and fibrils. In sporadic AD, Aβ oligomers (especially those derived from the 42-amino acid long isoform, Aβ42) are believed to be particularly harmful, whereas fibrils appear to represent an aggregation endpoint that may be relatively benign.

Peptide backbone conformations can be altered through introduction of D-amino acids (“Chiral Editing”), and replacement of L- by D-amino acids across the entire peptide yields mirror image (“D-“)Aβ. Because of the enantiomeric relation, D-Aβ has to possess an identical oligomer-pro­to­fibril-fibril distribution to that of the natural (“L-“) stereoisomer. However, all 3D-structural para­meters are mirrored in D-Aβ42, including the peptide backbone. Through stereochemical argu­ments, we envisioned that racemic Aβ42 should exhibit increased fibril formation and reduced toxicity. We synthesized the two enantiomers of Aβ42 and found that their equimolar mixture exhibited pronounced acceleration of fibril formation, as compared to the enantiopure coun­ter­parts. This led to substantial suppression of oligomer formation and inhibition of toxicity in model cell-based systems. We termed this the “Chiral Inactivation” effect. The underlying molecular mechanisms that lead to the differences in biophysical and biological properties observed between enantiopure and racemic Aβ42 remain subject of active research in our laboratory.

Density functional theory (DFT) calculations on β-strand segments identified by solid state NMR indicate an energetic preference for antiparallel alignments between neighboring D and L molecules within rippled sheets of D,L-Aβ fibrils.

Jevgenij A. Raskatov, Alejandro R. Foley, John M. Louis, Wai-Ming Yau, Robert Tycko J. Am. Chem. Soc. 2021.

p3 Peptide Revisited: A Case for Amyloid-α

Despite the vast heterogeneity of amyloid plaques isolated from the brains of those with Alzheimer’s Disease (AD), the basis of the Amyloid Cascade Hypothesis targets a single peptide, the amyloid-β (Aβ) peptide. The countless therapeutic efforts targeting the production and aggregation of this specific peptide have been met with disappointment, leaving many to question the role of Aβ in AD. An alternative cleavage product of the Amyloid-β protein precursor, called the p3 peptide, which has also been isolated from the brains of AD patients, has been largely absent from most Aβ-related studies. Typically referred to as non-amyloidogenic and even suggested as neuroprotective, the p3 peptide has garnered little attention aside from some conflicting findings on cytotoxicity and potential self-assembly to form higher order aggregates.

By probing the self-assembly of the p3 peptide, we found that p3 aggregates to form oligomers and fibrils and, when compared with Aβ, displays enhanced aggregation rates. Our findings highlight the solubilizing effect of the N-terminus of Aβ and the favorable formation of structures formed through C-terminal hydrophobic peptide interfaces. Based on our findings, we suggest a reevaluation of the current therapeutic approaches targeting only the β-secretase pathway of AD, given that the α- secretase pathway is also amyloidogenic.

Amyloid-β vs. Amyloid-α: Fibril Formation & Kinetics

Left: TAMRA quenching assay. Right: ThT kinetic profile of p3 vs. Aβ(1-40). p3 ThT kinetic profile exhibits enhanced fibril formation and lacks characteristic “lag phase.”

Kuhn, A. J.; Abrams, B.S.; Knowlton, S.; Raskatov, J. A. ACS Chem. Neurosci. 2020.