Department of Chemistry, Binghamton University, Vestal, NY 13850, USA.
Small Scale Systems Integration and Packaging (S3IP), Binghamton University, Vestal, NY 13850, USA.
Biophys Chem. 2023 Jun;297:107008. doi: 10.1016/j.bpc.2023.107008. Epub 2023 Mar 21.
Non-specific disruption of cellular membranes induced by aggregation of exogeneous β-amyloid (Aβ) 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 Aβ aggregates. Yet, the feasibility of in-cell ssNMR spectroscopy to probe Aβ-membrane interactions in native cellular environments has rarely been tested. Here we report the application of in-cellP 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 spectroscopy and P spin-spin (T) relaxation measurements. The P T 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 μM 40-residue Aβ (Aβ) peptides, the P T relaxation is instantly accelerated. This work demonstrates the feasibility of using in-cellP ssNMR to investigate the Aβ-membrane interactions in the biologically relevant cellular system.
外源性β-淀粉样蛋白（Aβ）肽聚集诱导的细胞膜非特异性破坏被认为是阿尔茨海默病（AD）的一种可行的病理机制。固态核磁共振（ssNMR）光谱学已广泛应用于模型脂质体，为研究膜与 Aβ 聚集体之间的分子相互作用提供了重要的见解。然而，在细胞内探测 Aβ-膜相互作用的固态 NMR 光谱学的可行性在天然细胞环境中很少得到测试。在这里，我们报告了在中度魔角旋转（MAS）条件下，对活的小鼠成神经细胞瘤 Neuro-2a（N2a）细胞进行细胞内 P ssNMR 光谱学的应用。在 277 K 下，5 kHz MAS 频率下，细胞活力和细胞质膜完整性可保持长达 6 小时，这允许进行直接极化 P 光谱和 P 自旋-自旋（T）弛豫测量。与用可比主要磷脂组成制备的模型脂质体相比，N2a 细胞的 P T 弛豫时间常数显著增加。加入 5 μM 40 残基 Aβ（Aβ）肽后，P T 弛豫立即加速。这项工作证明了使用细胞内 P ssNMR 研究在生物学相关细胞系统中 Aβ-膜相互作用的可行性。
