In-cell 31P solid-state NMR measurements of the lipid dynamics and influence of exogeneous β-amyloid peptides on live neuroblastoma neuro-2a cells

Non-specific disruption of cellular membranes induced by aggregation of exogeneous beta-amyloid (A beta) peptides is considered a viable pathological mechanism in Alzheimer's disease (AD). The solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been widely applied in model liposomes to provide important insights on the molecular interactions between membranes and A beta aggregates. Yet, the feasibility of in-cell ssNMR spectroscopy to probe A beta-membrane interactions in native cellular environments has rarely been tested. Here we report the application of in-cell P-31 ssNMR spectroscopy on live mouse neuroblastoma Neuro-2a (N2a) cells under moderate magic angle spinning (MAS) conditions. Both cell viability and cytoplasmic membrane integrity are retained for up to six hours under 5 kHz MAS frequency at 277 K, which allow applications of direct-polarization P-31 spectroscopy and P-31 spin-spin (T-2) relaxation measurements. The P-31 T-2 relaxation time constant of N2a cells is significantly increased compared with the model liposome prepared with comparable major phospholipid compositions. With the addition of 5 mu M 40-residue A beta (A beta(1-40)) peptides, the P-31 T-2 relaxation is instantly accelerated. This work demonstrates the feasibility of using in-cell P-31 ssNMR to investigate the A beta-membrane interactions in the biologically relevant cellular system.