Aβ-42 sidechain deamidation at Q15 and N27 modulate protein aggregation and microglial responses via altered cytokine production and CD68 expression

The progressive aggregation of amyloid beta (Aβ) monomers into oligomers is a critical factor in Alzheimers disease (AD) pathogenesis. Although mutated forms of Aβ have been shown to display altered aggregation dynamics, the specific effects of deamidated Aβ on microglial function remain understudied. Our research group previously found that the deamidated variant Aβ-42-N27D modified Aβ aggregation, reduced neurotoxicity, and reduced microglial reactivity, but the impact of Aβ-42 side chain deamidation in general on such parameters remained unclear. Here, we expanded on our prior work by investigating how two site-specific Aβ-42 mutations (Q15E & N27D), where neutral amide side chains are replaced with negatively charged carboxylic acids, affect aggregation and microglial immune response using a mouse microglial cell line. Size exclusion chromatography revealed that Aβ-42-Q15E and Aβ-42-N27D exhibit distinct aggregation profiles compared to Aβ-42 wild type (WT). Multiplexed analysis of 8 cytokines secreted into the culture medium revealed that Aβ-42-Q15E and Aβ-42-N27D decrease the expression of inflammatory cytokines such as IL-6, IP-10, and MIP-1α relative to Aβ-42-WT. Immunocytochemistry revealed that Aβ-42-Q15E and Aβ-42-N27D decrease CD68 expression relative to Aβ-42-WT. These findings demonstrate that deamidation significantly alters Aβ-42 aggregation and microglial activation, suggesting structural modifications to Aβ-42 modulate inflammatory signaling in AD. This work provides a foundation for future studies on Aβ-42 post-translational modifications as potential therapeutic targets in AD.