Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling

The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.

神经系统实现长时程可塑性的能力，植根于细胞与突触蛋白质组的动态变化。尽管已有大量研究探索了可塑性过程中神经元蛋白质组的动态改变，但针对星形胶质细胞对应蛋白质组的研究关注度却相对有限。诚然，受技术难题制约，细胞类型特异性蛋白质组的鉴定工作进展缓慢。本研究基于生物正交氨基酸叠氮正亮氨酸（azidonorleucine）与突变型小家鼠甲硫氨酰-tRNA合成酶L274G（mutated Mus musculus methionyl-tRNA synthetaseL274G），开发了一套适用于哺乳动物共培养模型的细胞类型特异性代谢标记技术，可实现叠氮正亮氨酸掺入新生合成的蛋白质中。该技术在完成细胞类型特异性蛋白质标记的同时，可维持神经元与星形胶质细胞的细胞活性。此外，本研究成功对星形胶质细胞的新生合成蛋白质进行了标记，并在神经元-胶质细胞共培养模型的星形胶质细胞中，鉴定出经脑源性神经营养因子（Brain-derived neurotrophic factor）处理后上调的连接蛋白43（Connexin-43）与60S核糖体蛋白L10a（60S ribosomal protein L10a）。综上，本研究通过细胞类型特异性代谢标记技术，成功实现了星形胶质细胞蛋白质组与神经元蛋白质组的有效分离，为细胞类型特异性蛋白质组动态变化的分析提供了全新的研究路径。