(182x) Deamidation of Amino Acid Side-Chain in Alzheimer’s Amyloid Beta(1-42) Alters Aggregation Pathways and Reduce Toxic Oligomer Formation

Amyloid beta(Aβ) is an aggregation-prone peptide and a primary pathological hallmark of Alzheimer's disease, affecting over 55 million people worldwide. The 40 amino acid long isoform, Aβ40, is the most prevalent, but the less common 42 amino acid long isoform, Aβ42, is substantially more toxic. This is believed to be due to its higher aggregation propensity. While Aβ can exist in different aggregated forms, it is now believed that oligomeric intermediates are most toxic^[1], whereas fibrillary deposits, initially believed to be the culprit, are now considered relatively benign. These oligomers disrupt synaptic function, trigger inflammatory responses, and lead to neuronal death. Despite their critical role in disease pathogenesis, a significant knowledge gap exists regarding oligomer structure and formation mechanisms, limiting our ability to develop effective therapeutic interventions. The intermolecular salt bridge between Asp23-Lys28 has been identified as a key stabilizing element in Aβ42 fibrils, suggesting that targeted disruption of such electrostatic interactions could redirect aggregation pathways away from toxic oligomer formation. Building on previous work by Foley et al^[2]., who demonstrated that single amino acid chiral substitution from L-Serine to D-Serine on S26 can dramatically alter aggregation kinetics and toxicity profiles, we hypothesized that strategic deamidation mutations could provide a novel approach to attenuate oligomer formation without eliminating the peptide's physiological functions.

We investigated how the deamidation of Aβ42 (N27D and Q15E) impacts peptide stability and aggregation pathways. These specific sites were selected based on molecular modeling predictions of their potential to disrupt critical stabilizing interactions within the peptide structure. Using thioflavin T (ThT) fluorescence assays, we monitored aggregation kinetics of wild-type (WT) and mutant peptides under physiologically relevant conditions (pH 7.4, 37°C). Size-exclusion chromatography was employed to quantitatively analyze oligomer formation profiles at various time points during the aggregation process. Additionally, we conducted cytokine expression studies using BV-2 microglial cells exposed to both monomeric and oligomeric species to evaluate the inflammatory response as an indirect measure of toxicity between WT and mutant forms. We conducted all atom molecular dynamics (MD) simulations with enhanced sampling for WT and N27D monomers to evaluate the D23-K28 salt-bridge contact and disruption. ¹H Solution NMR is used to study the monomeric transition to higher molecular aggregates by measuring the signal decay and spectral width broadening over time.

Compared to wild-type Aβ42, the N27D mutant exhibited a significantly extended lag phase in fibril formation, with ThT fluorescence showing approximately 3-fold longer time to reach the exponential growth phase. This extended kinetic profile suggests an altered aggregation pathway. Size-exclusion chromatography revealed that while WT Aβ42 readily formed low-to-high molecular weight aggregates (oligomers), the N27D mutant formed these species at a substantially reduced rate, with approximately 70% fewer oligomers detected at equivalent time points. Our cytokine studies demonstrated that both oligomers and monomers of N27D maintained cytokine expression comparable to WT counterparts on a per-molecule basis. However, the significantly reduced oligomer formation by N27D suggests an overall diminished inflammatory response, indirectly indicating a lower toxicity profile. To further investigate the role of deamidation, we examined the Q15E mutation, which introduces potential electrostatic interactions with K16. Remarkably, Q15E showed a size-exclusion chromatography profile highly similar to N27D, with significantly reduced oligomer formation compared to WT Aβ42.

Our results confirm that strategic deamidation mutations affecting electrostatic interactions cause Aβ42 to follow alternative aggregation pathways, reducing the formation of toxic oligomeric species. These findings provide new insights into the molecular mechanisms governing Aβ42 aggregation and establish deamidation as a potential strategy for modulating this process. This approach represents a different approach from traditional Aβ-targeting strategies that focus on complete clearance or inhibition of all aggregates. Instead, our work suggests that selective modification of aggregation pathways could reduce toxicity while potentially preserving physiological functions of the peptide. Future studies will explore whether these deamidation-inspired modifications could serve as the basis for designing enzymes that specifically deamidate the amino acids, potentially offering new therapeutic avenues for Alzheimer's disease and related proteinopathies.

References:

[1] Haass, C.; Selkoe, D. J. Soluble Protein Oligomers in Neurodegeneration: Lessons from the Alzheimer’s Amyloid β-Peptide. Nat Rev Mol Cell Biol 2007, 8 (2), 101–112. https://doi.org/10.1038/nrm2101.

[2] Foley, A. R.; Finn, T. S.; Kung, T.; Hatami, A.; Lee, H.-W.; Jia, M.; Rolandi, M.; Raskatov, J. A. Trapping and Characterization of Nontoxic Aβ42 Aggregation Intermediates. ACS Chem. Neurosci. 2019, 10 (8), 3880–3887. https://doi.org/10.1021/acschemneuro.9b00340.