Astroglial Orexin in Sleep Disorders

描述(由申请人提供)：我们在两个已建立的发作性睡病动物模型中首次成功地使用神经元增食欲素基因转移来改善发作性睡病的症状。这种影响是特定部位的，并依赖于代理神经元的连接性。由于神经元的增食欲素可以在多个远端部位分泌，其中一些可能调节发作性睡病的行为，而另一些则不能，我们提出了一种通过在星形胶质细胞中表达增食欲素来定位基因传递的方法。星形胶质细胞-食欲素将在已经包含食欲素受体的局部区域重新建立配体-受体联系。星形胶质增食欲素基因传递将作为一种经济有效的神经生物学工具来理解睡眠背后的网络。在目标1中，将增食欲素基因插入到星形胶质细胞(GFAP启动子驱动)中的三个特定脑区，其中两个与网络睡眠模型(TMN，Pons)有关，第三个纹状体作为对照。我们在增食欲素-阿塔辛-3小鼠模型中的初步结果表明，在TMN中，胶质增食欲素(rAAV-GFAP-增食欲素)可以完全挽救清醒状态，但不会导致猝倒。在脑桥中，它可以减少78%的晕厥，但不会醒着。因此，存在神经胶质增食欲素的部位特异性。在目标1中，星形胶质细胞食欲素将由光遗传刺激驱动，以确定这种刺激是否进一步刺激觉醒或阻断猝倒，特别是在目标1中无效的部位。建议进行适当对照的实验，包括食欲素受体拮抗剂，以加强这一结论。体外钙成像研究将确定基因插入的ChR2受体的功能。据我们所知，这是星形胶质增食欲素在睡眠障碍中的首次应用。基因转移是非常重要的 性价比高，已用于临床，并将证明遗传药理学可以用于 阐明睡眠障碍中的网络。

DESCRIPTION (provided by applicant): We have demonstrated the first successful use of neuronal-orexin gene transfer to ameliorate symptoms of narcolepsy in two established animal models of the disease. The effects were site specific and depended on the connectivity of the surrogate neurons. Since neuronal orexin can be secreted at multiple distal sites, some of which may regulate narcoleptic behavior whereas others do not, we propose a localized gene delivery method by expressing orexin in astroglia. Astroglial-orexin will re-establish the ligand-receptor link in a localized area that already contains the orexin receptors. Astroglial-orexin gene deliver will serve as a cost-effective neurobiological tool to understand the networking underlying sleep. In aim 1 the gene for orexin will be inserted into astroglia (GFAP promoter driven) in three specific brain regions, two of which are implicated in network sleep models (TMN, pons) and the third, striatum, will serve as control. Our preliminary results in the orexin-ataxin-3 mice model indicate that in the TMN glial-orexin (rAAV-GFAP-orexin) completely rescues waking but not cataplexy. In the pons it produces a 78% reduction in cataplexy but not waking. Thus, there is site- specificity of the glial-orexin. In aim 2 astroglial-orexin will be driven by optogenetic stimulation to determine whether such stimulation further stimulates waking or blocks cataplexy, especially in ineffective sites in aim 1. Experiments with appropriate controls, including orexin receptor antagonist are proposed to strengthen the conclusions. In-vitro calcium imaging study will determine functionality of the genetically inserted ChR2 receptors. To the best of our knowledge this is the first use of astroglial-orexin in sleep disorders. Gene transfer is extremely cost-efficient, it is used clinically and it will demonstrate that genetic pharmacology can be used to elucidate the network in sleep disorders.