Turku Centre for Biotechnology, University of Turku , FI-20014 Turku, Finland.
Institute of Biomedicine, University of Helsinki , FI-00014 Helsinki, Finland.
ACS Chem Neurosci. 2016 Jun 15;7(6):749-56. doi: 10.1021/acschemneuro.6b00002. Epub 2016 Apr 28.
Anesthetics are widely used in medical practice and experimental research, yet the neurobiological basis governing their effects remains obscure. We have here used quantitative phosphoproteomics to investigate the protein phosphorylation changes produced by a 30 min isoflurane anesthesia in the adult mouse hippocampus. Altogether 318 phosphorylation alterations in total of 237 proteins between sham and isoflurane anesthesia were identified. Many of the hit proteins represent primary pharmacological targets of anesthetics. However, findings also enlighten the role of several other proteins-implicated in various biological processes including neuronal excitability, brain energy homeostasis, synaptic plasticity and transmission, and microtubule function-as putative (secondary) targets of anesthetics. In particular, isoflurane increases glycogen synthase kinase-3β (GSK3β) phosphorylation at the inhibitory Ser(9) residue and regulates the phosphorylation of multiple proteins downstream and upstream of this promiscuous kinase that regulate diverse biological functions. Along with confirmatory Western blot data for GSK3β and p44/42-MAPK (mitogen-activated protein kinase; reduced phosphorylation of the activation loop), we observed increased phosphorylation of microtubule-associated protein 2 (MAP2) on residues (Thr(1620,1623)) that have been shown to render its dissociation from microtubules and alterations in microtubule stability. We further demonstrate that diverse anesthetics (sevoflurane, urethane, ketamine) produce essentially similar phosphorylation changes on GSK3β, p44/p42-MAPK, and MAP2 as observed with isoflurane. Altogether our study demonstrates the potential of quantitative phosphoproteomics to study the mechanisms of anesthetics (and other drugs) in the mammalian brain and reveals how already a relatively brief anesthesia produces pronounced phosphorylation changes in multiple proteins in the central nervous system.
麻醉剂在医学实践和实验研究中被广泛应用，但控制其效果的神经生物学基础仍然不清楚。我们使用定量磷酸蛋白质组学来研究 30 分钟异氟烷麻醉在成年小鼠海马体中产生的蛋白质磷酸化变化。总共在假手术和异氟烷麻醉之间鉴定出 237 种蛋白质中总共 318 种磷酸化改变。许多命中蛋白代表麻醉剂的主要药理学靶标。然而，这些发现还揭示了其他几种蛋白质的作用-涉及神经元兴奋性、大脑能量稳态、突触可塑性和传递以及微管功能等各种生物学过程-作为麻醉剂的潜在（次要）靶标。特别是，异氟烷增加糖原合酶激酶-3β（GSK3β）在抑制性 Ser（9）残基上的磷酸化，并调节该多功能激酶下游和上游的多种蛋白质的磷酸化，这些蛋白质调节各种生物学功能。除了 GSK3β和 p44/42-MAPK（有丝分裂原激活蛋白激酶；激活环磷酸化减少）的Western blot 数据验证外，我们还观察到微管相关蛋白 2（MAP2）在残基（Thr（1620、1623））上的磷酸化增加，这已经显示出其从微管上解离以及微管稳定性的改变。我们进一步证明，不同的麻醉剂（七氟醚、尿烷、氯胺酮）在 GSK3β、p44/p42-MAPK 和 MAP2 上产生与异氟烷观察到的基本相似的磷酸化变化。总的来说，我们的研究表明定量磷酸蛋白质组学在研究哺乳动物大脑中麻醉剂（和其他药物）的机制方面具有潜力，并揭示了相对短暂的麻醉如何在中枢神经系统中的多种蛋白质中产生明显的磷酸化变化。
