泛素蛋白连接酶MDM2在麻醉药物致发育大脑认知功能障碍中的作用及功能研究

Several studies have shown that sevoflurane anesthesia can impair synaptic function, leading to learning and memory impairment in young mice, but the underling mechanism is still unknown. The ubiquitin proteasome pathway is closely related to learning and memory. Our previous experiments revealed that the interaction of E3-linked enzyme Mouse double minute 2 homolog (MDM2) and postsynaptic dense protein-95 (PSD-95) were increased by sevoflurane anesthesia, which consequently induce the degradation of PSD-95 and the changes of synaptic plasticity. MDM2 inhibitor Nutline-3 could attenuate learning and memory impairment induced by sevoflurane anesthesia. Our project will systematically determine the role of MDM2 in general anesthetics-induced cognitive impairment in young mice. We will determine how general anesthetics affect MDM2 by using in vivo and in vitro experiments, and using the technique of a protein purification approach combined with high-performance liquid chromatography/tandem mass spectrometry to carry out a system-wide screening of MDM2 substrates and the interaction proteins that influence the function of MDM2. We will clarify the specific regulation mechanism of general anesthetics-induced learning and memory impairment and identify the possibility of MDM2 as a new target to protect general anesthetics-induced learning and memory impairment. Our goal is to use general anesthetics as a research tool to elucidate the effect of ubiquitin protease regulation pathway (such as MDM2/PSD-95 pathway) on the development of cognitive function in young mice. This is the first time we explain the role of E3 ligase in the mechanism of general anesthetics-induced brain neurotoxicity.

七氟醚多次麻醉导致发育大脑学习记忆功能损伤，机制尚不明确。泛素蛋白酶体通路与学习记忆密切相关。申请人发现七氟醚麻醉促进泛素蛋白连接酶E3小鼠双微体基因2（MDM2）与突触后致密蛋白-95（PSD-95）的结合，导致其泛素化降解增加和突触功能的改变。MDM2抑制剂Nutline-3可改善七氟醚麻醉引起的学习记忆损伤。本课题拟进一步系统确定MDM2在麻醉药物（七氟醚/异丙酚）致发育大脑认知功能障碍中的作用；通过体内、外实验及蛋白质纯化、高效液相色谱/串联质谱等技术考察多次麻醉中MDM2的调控机制，发掘MDM2在神经系统的新底物及影响MDM2功能的相互作用蛋白；阐明麻醉药物致学习记忆损伤的具体调控机制和MDM2作为预防靶点的可能性。本研究应用麻醉药作为研究工具来阐明泛素蛋白酶调控（如MDM2/PSD-95降解通路）对发育大脑认知功能的影响，创新性从E3连接酶角度探讨麻醉药物发育大脑神经毒性机制。

麻醉药物多次作用会影响大脑学习记忆功能损伤，机制尚不明确。课题组前期发现七氟醚麻醉促进泛素蛋白连接酶E3小鼠双微体基因2（MDM2）与突触后致密蛋白-95（PSD-95）的结合，导致其泛素化降解增加和突触功能的改变。MDM2抑制剂Nutline-3可改善七氟醚麻醉引起的学习记忆损伤。进一步通过质谱分析，鉴定出MDM2的相互作用的蛋白为FBXO22，随后进行免疫共沉淀实验验证，因为目前抗体的质量原因，只能单向验证MDM2和FBXO22的相互作用。同时发现过表达MDM2的神经元，细胞数明显减少，考虑过表达MDM2与凋亡通路相关。因为本课题主要研究突触可塑性和学习记忆障碍，故将研究重点放在探讨泛素蛋白连接酶E3——FBXO22在神经系统的功能。课题组研究发现较高剂量异丙酚（100mg/kg）多次作用后FBXO22表达增加， NMDAR1和PSD-95蛋白水平降低，伴随学习记忆障碍。进一步降低FBXO22的表达后，发现改善了异丙酚多次作用引起的谷氨酸能突触可塑性改变和学习记忆障碍。提示FBXO22是异丙酚多次作用引起的学习记忆障碍的潜在靶点。我们进一步拓展了研究内容，认知功能障碍治疗过程中血脑屏障限制了大部分药物向脑部的转运，鼻腔给药途径通过天然解剖结构可以直接将药物输送到大脑，而没有通过全身吸收，从而避免了副作用，并极大的提高了药物在脑部的浓度和治疗功效。我们的运用纳米材料包裹亮氨酸重复激酶2(LRRK2)，通过鼻腔递送，达到缓释、精准递送的目的，将LRRK2抑制剂递送至大脑，协同作用减轻小胶质细胞的炎症反应并通过体内、体外和行为学实验均验证可以改善脓毒血症引起的认知障碍。