TRPV4介导的星形胶质细胞体积改变参与睡眠-觉醒的调控机制

Treatment of sleep disorders is different in clinical practice. Breakthrough progress of the treatment would rely on further exploration of sleep-wakefulness mechanism. Neurons releasing sleep/wake-inducing neurotransmitters were regarded as the core of sleep-wake mechanism in traditional theory. However, updated findings suggested that, astrocytic regulation of sleep-wakefulness is important. Astrocytes effectively regulate cell volume and extracellular microenvironment and astrocytic calcium signaling is sleep-wakefulness state dependent. Applicant previously reported that,alterations of extracellular cation compositions could shift sleep-wakefulness states.Our preliminary data suggested that, arousal stimulus expand astrocytic cell volume, while sleep-inducing interventions shrink the cell volume, which mediates state-dependent changes in extracellular microenvironment. Transient receptor potential vanilloid 4(TRPV4)is an irreplaceable calcium channel involved in astrocytic volume regulation. We proposed that, TRPV4-mediated astrocytic volume changes regulate extracellular microenvironment and involve in sleep-wakefulness states control. This study would use in vitro and in vivo methods to manipulate astrocytic TRPV4 expression and function, and observe if it affect sleep-wakefulness state-dependent astrocytic volume changes and intracellular calcium signaling, as well as sleep-wake cycle. This study could potentially provide novel therapeutic target for sleep disorders.

睡眠障碍的治疗是临床难题，突破性进展有待于对睡眠-觉醒机制的深入挖掘。既往研究认为，释放促眠或促醒神经递质的神经元是其核心，而近年来研究显示，星形胶质细胞参与了睡眠-觉醒调控。星形胶质细胞可高效调节细胞体积与细胞外微环境，且其细胞内钙信号具有睡眠-觉醒状态依赖性的特点。申请人已发表研究示，改变细胞外离子浓度组合可调控睡眠-觉醒状态。前期数据示，星形胶质细胞在觉醒刺激下肿胀，而在睡眠诱导下缩小，可改变局部细胞外微环境。瞬时感受器电位离子通道（TRPV4）是介导星形胶质细胞体积改变的重要钙离子通道。我们提出假设，TRPV4介导的星形胶质细胞体积改变，可调节细胞外微环境，参与睡眠-觉醒状态的转换与维持。本研究将特异性调控TRPV4表达及功能，在体和离体对比观察睡眠-觉醒状态相关的星形胶质细胞体积与胞内钙信号变化，分析睡眠-觉醒周期的改变。这一研究将为睡眠-觉醒相关疾病提供新的治疗靶点。

睡眠-觉醒调控机制是研究热点。既往研究以神经元为中心，已有重要发现。申请人关注科学问题：星形胶质细胞在睡眠-觉醒调控中的作用与机制？申请人前期报道了细胞外间隙离子微环境可正向调控睡眠-觉醒状态。而脑内星形胶质细胞是重要的离子稳态维护者。遂提出科学假设：星形胶质细胞可通过调控细胞外微环境离子环境而影响睡眠觉醒状态的转换与维持。研究围绕瞬时感受器电位离子通道(TRPV4)进行特异性分子调控，TRPV4可灵敏感知渗透压、膜表面张力改变，且与钾、氯及水通道存在共定位和功能偶联。本研究首先运用双光子、宽视场单光子等在体成像技术特异性标记并展现了在清醒-麻醉-麻醉撤药不同皮层状态下，星形胶质细胞体积在麻醉诱导后发生可逆性缩小；细胞内钙信号与细胞体积改变的时相存在对应关系。在体电生理数据显示，细胞外间隙容积在呈现麻醉时扩大而撤药后恢复的动态改变。本部分数据证实了我们的猜想，星形胶质细胞在清醒-麻醉（睡眠）状态下可发生结构与功能的动态改变。我们构建了KO与fl/fl小鼠品系，以探讨TRPV4对皮层状态调控的重要性。经过两轮构建，我们已获得了敲除外显子3-16片段的杂合小鼠与fl/fl小鼠品系。但在繁育过程中，未能获得TRPV4纯合敲除小鼠，以及星形胶质细胞特异性TRPV4 敲除小鼠，提示TRPV4可能不可完全缺失。现有数据显示，杂合敲除鼠与同笼对照在睡眠-觉醒表型、认知与情绪、运动等行为表型中，不具有统计学差异。经侧脑室给药，我们并未发现TRPV4特异性调控剂对脑电波谱信号的显著性改变。然而，我们运用星形胶质细胞特异性（Aldh1l1-cre）病毒进行脑区TRPV4敲除，已取得初步数据，将被进一步验证。本研究的意义在于指出了星形胶质细胞在睡眠（麻醉）-觉醒调控机制中或占据重要作用，其调控脑内细胞外微环境介导皮层状态转换的相关分子机制或为新型睡眠改善药物的发现提供新靶点和新思路。