硅涂层磁性纳米颗粒激活小胶质细胞并诱导神经毒性 D-丝氨酸分泌。

BACKGROUND: Nanoparticles have been studied for brain imaging, diagnosis, and drug delivery owing to their versatile properties due to their small sizes. However, there are growing concerns that nanoparticles may exert toxic effects in the brain. In this study, we assessed direct nanotoxicity on microglia, the resident macrophages of the central nervous system, and indirect toxicity on neuronal cells exerted by silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO(RITC)]. METHODS: We investigated MNPs@SiO(RITC)-induced biological changes in BV2 murine microglial cells via RNA-sequencing-based transcriptome analysis and gas chromatography-mass spectrometry-based intracellular and extracellular amino acid profiling. Morphological changes were analyzed by transmission electron microscopy. Indirect effects of MNPs@SiO(RITC) on neuronal cells were assessed by Transwell-based coculture with MNPs@SiO(RITC)-treated microglia. MNPs@SiO(RITC)-induced biological changes in the mouse brain in vivo were examined by immunohistochemical analysis. RESULTS: BV2 murine microglial cells were morphologically activated and the expression of Iba1, an activation marker protein, was increased after MNPs@SiO(RITC) treatment. Transmission electron microscopy analysis revealed lysosomal accumulation of MNPs@SiO(RITC) and the formation of vesicle-like structures in MNPs@SiO(RITC)-treated BV2 cells. The expression of several genes related to metabolism and inflammation were altered in 100 µg/ml MNPs@SiO(RITC)-treated microglia when compared with that in non-treated (control) and 10 µg/ml MNPs@SiO(RITC)-treated microglia. Combined transcriptome and amino acid profiling analyses revealed that the transport of serine family amino acids, including glycine, cysteine, and serine, was enhanced. However, only serine was increased in the growth medium of activated microglia; especially, excitotoxic D-serine secretion from primary rat microglia was the most strongly enhanced. Activated primary microglia reduced intracellular ATP levels and proteasome activity in cocultured neuronal cells, especially in primary cortical neurons, via D-serine secretion. Moreover, ubiquitinated proteins accumulated and inclusion bodies were increased in primary dopaminergic and cortical neurons cocultured with activated primary microglia. In vivo, MNPs@SiO(RITC), D-serine, and ubiquitin aggresomes were distributed in the MNPs@SiO(RITC)-treated mouse brain. CONCLUSIONS: MNPs@SiO(RITC)-induced activation of microglia triggers excitotoxicity in neurons via D-serine secretion, highlighting the importance of neurotoxicity mechanisms incurred by nanoparticle-induced microglial activation.

背景：由于其小尺寸带来的多种特性，纳米颗粒已被用于脑成像、诊断和药物输送研究。然而，人们越来越担心纳米颗粒可能会对大脑产生毒性作用。在这项研究中，我们评估了载有罗丹明 B 异硫氰酸酯染料 [MNPs@SiO（RITC）] 的二氧化硅涂层磁性纳米颗粒对小胶质细胞（中枢神经系统的固有巨噬细胞）的直接纳米毒性以及对神经元细胞的间接毒性。

方法：我们通过基于 RNA 测序的转录组分析和基于气相色谱-质谱的细胞内和细胞外氨基酸谱分析，研究了 MNPs@SiO（RITC）诱导的 BV2 小鼠小胶质细胞的生物学变化。通过透射电子显微镜分析了形态变化。通过用 MNPs@SiO（RITC）处理的小胶质细胞进行 Transwell 共培养，评估了 MNPs@SiO（RITC）对神经元细胞的间接影响。通过免疫组织化学分析研究了 MNPs@SiO（RITC）在体内诱导的小鼠脑生物学变化。

结果：BV2 小鼠小胶质细胞形态激活，MNPs@SiO（RITC）处理后 Iba1（一种激活标志物蛋白）的表达增加。透射电子显微镜分析显示 MNPs@SiO（RITC）的溶酶体积累和 MNPs@SiO（RITC）处理的 BV2 细胞中囊泡样结构的形成。与未处理（对照）和 10 µg/ml MNPs@SiO（RITC）处理的小胶质细胞相比，100 µg/ml MNPs@SiO（RITC）处理的小胶质细胞中几种与代谢和炎症相关的基因表达发生改变。联合转录组和氨基酸谱分析表明，包括甘氨酸、半胱氨酸和丝氨酸在内的丝氨酸家族氨基酸的转运增强。然而，仅在激活的小胶质细胞的生长培养基中观察到丝氨酸增加；特别是，原代大鼠小胶质细胞中兴奋性 D-丝氨酸的分泌增加最为明显。激活的原代小胶质细胞通过 D-丝氨酸分泌降低共培养神经元细胞（尤其是原代皮质神经元）中的细胞内 ATP 水平和蛋白酶体活性。此外，与激活的原代小胶质细胞共培养的原代多巴胺能和皮质神经元中积累了泛素化蛋白并增加了包涵体。在体内，MNPs@SiO（RITC）、D-丝氨酸和泛素聚集体在 MNPs@SiO（RITC）处理的小鼠脑中分布。

结论：MNPs@SiO（RITC）诱导的小胶质细胞激活通过 D-丝氨酸分泌引发神经元的兴奋性毒性，突出了纳米颗粒诱导的小胶质细胞激活引起的神经毒性机制的重要性。