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

Griffin, M. N.; Dinakarapandian, D. M.; Li, C.; Ramaswamy, P.; Saheba, S. J.; Lee, J. C.; Sudarshan, T. R.; Sajimon, M.; Wheeler, C. J.; Raskatov, J. A.; Paravastu, A. K.; Wood, L. B.

doi:10.1101/2025.10.22.684043

bioRxiv · 10.1101/2025.10.22.684043

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

Griffin, M. N.·Dinakarapandian, D. M.·Li, C.·Ramaswamy, P.·Saheba, S. J.·Lee, J. C.·Sudarshan, T. R.·Sajimon, M.·Wheeler, C. J.·Raskatov, J. A.·Paravastu, A. K.·Wood, L. B.

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Abstract

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

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Griffin, M. N., Dinakarapandian, D. M., Li, C., Ramaswamy, P., Saheba, S. J., Lee, J. C., Sudarshan, T. R., Sajimon, M., Wheeler, C. J., Raskatov, J. A., Paravastu, A. K., Wood, L. B.. 2025-10-23. Aβ-42 sidechain deamidation at Q15 and N27 modulate protein aggregation and microglial responses via altered cytokine production and CD68 expression. https://doi.org/10.1101/2025.10.22.684043

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