海马LXRβ调控泛素化酶Nedd4-1参与睡眠剥夺所致学习记忆障碍的机制研究

Sleep deprivation (SD) causes learning and memory impairment by inducing neuroplasticity injury, which has become a prominent issue in affecting human physical and mental health. It is demonstrated that the ubiquitination enzymes Nedd4-1 participates in the learning and memory impairment in various models, and its mechanism is related to the down-regulation of the glutamate receptor AMPAR subunit GluA1. However, it is not clear that whether the molecular mechanism Nedd4-1 participates in sleep deprivation-induced learning and memory impairment. Our preliminary data showed that expression of LXRβ was decreased and expression of Nedd4-1 was increased in SD mice hippocampus, suggesting LXRβ may be a key transcription regulator of the Nedd4-1 expression, participating in GluA1 ubiquitination. This project intends to elucidate the role of activating LXRβ in down-regulating the expression of Nedd4-1, which inhibits the ubiquitination of GluA1 and improves sleep deprivation-induced learning and memory impairment. And we used electrophysiology, molecular biology and behavioral methods to explore underlying molecular mechanism of LXRβ on SD-induced learning and memory impairment. The research results will further uncover the pathological mechanism of SD-induced learning and memory impairment and provide evidence for new potential therapy targets.

睡眠剥夺（SD）诱发神经突触可塑性损伤，导致学习记忆障碍，已成为威胁公众健康的突出问题。在多个模型中已证实，泛素化酶Nedd4-1参与学习记忆障碍的发生，其机制与下调谷氨酸受体AMPAR亚基GluA1有关。但在睡眠剥夺所致学习记忆障碍中，Nedd4-1是否参与及其分子调控机制尚不明确。我们前期研究发现，睡眠剥夺小鼠中海马核受体LXRβ表达下降而Nedd4-1表达升高，提示LXRβ可能是调控Nedd4-1表达诱导GluA1泛素化的关键调节因子。本项目拟从表观遗传学角度阐明激活LXRβ下调Nedd4-1的表达，抑制GluA1的泛素化，进而改善睡眠剥夺所致学习记忆障碍的思路入手，结合电生理学、分子生物学和行为学方法，探讨LXRβ对Nedd4-1的转录调控，介导GluA1泛素化参与睡眠剥夺所致学习记忆障碍的影响，旨在为揭示睡眠剥夺所致学习记忆障碍的分子机制和发掘药物作用新靶点提供新思路和理论依据。

本课题是针对睡眠剥夺所致学习记忆障碍的机制研究，初步阐明了LXRβ参与Nedd4-1的表达调控，诱导AMPAR亚基GluA1泛素化参与睡眠剥夺诱导的学习记忆障碍的作用机制。课题以LXRβ功能异常参与睡眠剥夺导致的学习记忆障碍的为切入点，采用免疫荧光、免疫沉淀、蛋白印迹、病毒感染、激光共聚焦、电生理、行为学检测及药物干预等方法探讨LXRβ调控Nedd4-1转录参与睡眠剥夺诱导的学习记忆障碍的分子机制。我们的研究发现，睡眠剥夺小鼠中海马核受体LXRβ表达下降，提示LXRβ参与睡眠剥夺所致学习记忆障碍的发生，继而我们用shRNA敲低LXRβ的表达，结果发现GW3965可特异性激动LXRβ改善睡眠剥夺导致的学习记忆障碍。接着我们通过检测AMPAR亚基GluA1与GluA2的膜蛋白和总蛋白的表达水平，及高尔基染色、电生理检测睡眠剥夺对突触可塑性的影响，结果证明GW3965（10mg/kg, ip）逆转SD小鼠GluA1与GluA2的膜蛋白和总蛋白的表达，增加树突棘的数目，促进兴奋性突触传递从而改善睡眠剥夺所致的学习记忆障碍。此外，我们发现Nedd4-1表达升高在SD小鼠中，并利用新型的DNA-蛋白质互作研究方法Cleavage Under Target & Tagmentation（CUT&Tag）检测LXRβ与Nedd4-1的相互作用。本项证明了LXRβ可调控Nedd4-1的转录，介导GluA1泛素化参与睡眠剥夺所致学习记忆障碍的发生，为发掘药物新靶点提供理论依据。