Astroglial Orexin in Sleep Disorders

描述（由申请人提供）：我们已经证明了首次成功使用神经元-食欲素基因转移来改善两种已建立的疾病动物模型的发作性睡病症状。这种效果是位点特异性的，依赖于替代神经元的连通性。由于神经元食欲素可以在多个远端位点分泌，其中一些可能调节发作性睡行为，而另一些则没有，我们提出了一种通过在星形胶质细胞中表达食欲素的局部基因传递方法。星形胶质-食欲素将在已经含有食欲素受体的局部区域重新建立配体-受体联系。星形胶质-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了解睡眠背后的网络。在目标1中，食欲素基因将被插入到大脑三个特定区域的星形胶质细胞（GFAP启动子驱动）中，其中两个与网络睡眠模式（颞叶神经网络，脑桥）有关，而第三个纹状体将作为对照。我们在orexin-ataxin-3小鼠模型中的初步结果表明，TMN中的胶质-orexin （raav - gmap -orexin）完全恢复清醒，但不能恢复猝倒。在脑桥上，它能减少78%的猝倒，但不会使人清醒。因此，胶质-食欲素具有位点特异性。在目的2中，星形胶质-食欲素将被光遗传刺激驱动，以确定这种刺激是否会进一步刺激清醒或阻止发作，特别是在目的1中的无效部位。建议采用适当的对照实验，包括食欲素受体拮抗剂，以加强结论。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，这是星形胶质促食欲素在睡眠障碍中的首次应用。基因转移

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.