Stimulating microglial neuroinflammation with amyloid-beta oligomers compatible with solid-state NMR structural characterization

Dinakarapandian, D. M.; Rao Sudarshan, T.; Griffin, M. N.; Ramaswamy, P.; Nguyen, P. H.; Chiu, T.-Y.; Li, C.; Kamalaldinezabadi, S. S.; Watzlawik, J. O.; Rosenberry, T. L.; Stagg, S. M.; Wen, Z.; Wood, L. B.; Paravastu, A. K.

doi:10.64898/2026.10.14.759149

bioRxiv · 10.64898/2026.10.14.759149

Stimulating microglial neuroinflammation with amyloid-beta oligomers compatible with solid-state NMR structural characterization

Dinakarapandian, D. M.·Rao Sudarshan, T.·Griffin, M. N.·Ramaswamy, P.·Nguyen, P. H.·Chiu, T.-Y.·Li, C.·Kamalaldinezabadi, S. S.·Watzlawik, J. O.·Rosenberry, T. L.·Stagg, S. M.·Wen, Z.·Wood, L. B.·Paravastu, A. K.

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Abstract

Soluble amyloid-{beta} (A{beta}) oligomers are considered the primary neurotoxic species in Alzheimer's disease, yet the relationship between oligomer structure and biological activity remains poorly understood. Oligomer sample preparations amenable to high-resolution structural characterization have not been tested in cell-based assays, mostly because detergents used to drive oligomer formation are toxic to cells. Here, we describe a detergent-free protocol in which size-exclusion chromatography-isolated A{beta}42 monomers spontaneously oligomerize at 37 {degrees}C in a concentration- and temperature-dependent manner. Biophysical characterization shows a {beta}-sheet structure by circular dichroism, a self-associating population above 100 kDa by silver-stained SDS-PAGE, and bead-like strings by negative-stain TEM resembling the previously characterized SDS-induced 150 kDa oligomer. Solid-state NMR spectroscopy of selectively 13C/15N-labeled 37 {degrees}C oligomers yields well-resolved spectra with {beta}-strand chemical shifts, and two-dimensional 13C-13C DARR and CHHC experiments provide initial inter-residue distance constraints that reveal both similarities to and differences from the 150 kDa oligomer. Treatment of mouse microglial cells (MMCs) with 50 nM 37 {degrees}C oligomers elicited a robust pro-inflammatory cytokine response that exceeded that of monomeric A{beta}42, as demonstrated by Luminex multiplex profiling and principal component analysis. hiPSC-derived microglia (iMGs) treated with 1 {micro}M oligomers also separated from vehicle, establishing activity in a human cellular model. Monomeric A{beta}42 was inactive in both systems, indicating that inflammatory activity depends on the assembly state of A{beta}42 rather than on the presence of peptide alone. These results establish a detergent-free A{beta}42 oligomer preparation that is simultaneously compatible with solid-state NMR structural characterization and biologically active in neuroinflammation assays, providing a platform for studying microglial immune responses to oligomers.

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Dinakarapandian, D. M., Rao Sudarshan, T., Griffin, M. N., Ramaswamy, P., Nguyen, P. H., Chiu, T.-Y., Li, C., Kamalaldinezabadi, S. S., Watzlawik, J. O., Rosenberry, T. L., Stagg, S. M., Wen, Z., Wood, L. B., Paravastu, A. K.. 2026-10-20. Stimulating microglial neuroinflammation with amyloid-beta oligomers compatible with solid-state NMR structural characterization. https://doi.org/10.64898/2026.10.14.759149

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