Astroglial Orexin in Sleep Disorders

描述（由申请人提供）：我们已经在两种已建立的发作性睡病动物模型中首次成功地使用神经元-食欲素基因转移来改善发作性睡病的症状。这种效应具有部位特异性，并依赖于替代神经元的连接。由于神经元食欲素可以分泌在多个远端网站，其中一些可能会调节发作性睡眠行为，而其他人没有，我们提出了一个本地化的基因传递方法，通过表达食欲素在星形胶质细胞。星形胶质细胞-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-受体连接。星形胶质细胞-食欲素基因传递将作为一种具有成本效益的神经生物学工具来了解睡眠背后的网络。在目标1中，将食欲素的基因插入三个特定脑区域的星形胶质细胞（GFAP启动子驱动）中，其中两个涉及网络睡眠模型（TMN，脑桥），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，在TMN中，胶质-orexin（rAAV-GFAP-orexin）完全拯救清醒但不拯救昏迷。在脑桥中，它可以减少78%的昏迷，但不会醒来。因此，存在胶质-食欲素的位点特异性。在目标2中，星形胶质细胞-食欲素将由光遗传学刺激驱动，以确定这种刺激是否进一步刺激觉醒或阻断catabolism，特别是在目标1中的无效部位。建议使用适当对照（包括食欲素受体拮抗剂）进行实验，以加强结论。体外钙成像研究将确定基因插入的ChR 2受体的功能。据我们所知，这是第一次使用星形胶质细胞食欲素治疗睡眠障碍。基因转移是非常 成本效益，它是临床使用，它将证明，遗传药理学可以用于 来阐明睡眠障碍的网络。

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.