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

Soluble amyloid-β (Aβ) 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 Aβ42 monomers spontaneously oligomerize at 37 °C in a concentration- and temperature-dependent manner. Biophysical characterization shows a β-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 °C oligomers yields well-resolved spectra with β-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 °C oligomers elicited a robust pro-inflammatory cytokine response that exceeded that of monomeric Aβ42, as demonstrated by Luminex multiplex profiling and principal component analysis. hiPSC-derived microglia (iMGs) treated with 1 µM oligomers also separated from vehicle, establishing activity in a human cellular model. Monomeric Aβ42 was inactive in both systems, indicating that inflammatory activity depends on the assembly state of Aβ42 rather than on the presence of peptide alone. These results establish a detergent-free Aβ42 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.