The Brain's Valium: Investigating the Role of DBI in Regulating GABA-mediated Inhibition

项目概要： 苯二氮卓类药物（BZD）于20世纪50年代首次合成，是广泛使用的处方药， 通过结合GABA-A受体（GABAAR）而具有抗焦虑、肌肉松弛、镇静和抗惊厥作用， 大脑中主要的抑制性配体门控离子通道。自从他们合成以来，许多科学家都认为 大脑中存在一种内源性BZD，或叫内皮素1983年，一种候选多肽， 在GABAAR结合试验中，从脑匀浆中分离出置换的BZD。然而，证明这一点， 1 一种叫做安定结合抑制剂（DBI）的肽在大脑中缺乏功能性作用。 最近，使用DBI敲低实验证实，在外侧脑室下区， DBI及其肽片段之一ODN抑制GABA诱导的电流并促进 神经发生，表明DBI和ODN在体内作为GABAAR的负变构调节剂起作用。 2 然而，在丘脑网状核（在控制癫痫中起重要作用的核）中， 癫痫发作），DBI和/或DBI衍生肽被发现增强GABAAR电流并抑制癫痫发作 活动因此，根据大脑区域，DBI可以增强或抑制GABAAR活性。然而， 3 了解DBI如何实施这些行动。 来自单一蛋白质的产物如何对GABAAR电流产生正面与负面影响？做DBI- 衍生的肽，如ODN引起相同的功能效果DBI？不同的细胞类型是否能 处理不同的DBI肽？DBI是否经过后修饰（例如磷酸化、酰化）， 这些改变改变了DBI的生理效应拟议的实验将解决这些问题 基本问题，并将1）确定特定GABAAR亚单位组合是否介导阳性， 2）确定星形胶质细胞和神经元是否释放不同的DBI肽和/或 不同的后处理DBI，这影响其功能效应和3）确定星形胶质细胞是否 并且神经元表达不同的DBI转录变体。这些实验的成功完成将验证 DBI作为内源性BZD的作用，将为理解DBI如何调节GABA奠定基础。 介导的抑制作用。

Project Summary: First synthesized in the 1950s, benzodiazepines (BZDs) are widely prescribed drugs that exert their anxiolytic, muscle relaxant, sedative, and anticonvulsant actions by binding to GABA-A receptors (GABAARs), the main inhibitory ligand-gated ion channel in the brain. Since their synthesis, many scientists have theorized that there exists an endogenous BZD, or endozepine, in the brain. In 1983, a candidate polypeptide that displaced BZDs in a GABAAR binding assay was isolated from brain homogenates . However, proof that this 1 peptide called diazepam-binding inhibitor (DBI) had functional effects in the brain was lacking. Recently, using DBI knock-down experiments it was demonstrated in the subventricular zone of the lateral ventricles that DBI and one of its peptide fragments, ODN, inhibit GABA-induced currents and promote neurogenesis , indicating that DBI and ODN work as negative allosteric modulators of GABAARs in vivo. 2 However, in the thalamic reticular nucleus (a nucleus that plays an important role in controlling epileptic seizures), DBI and/or a DBI-derived peptide was found to enhance GABAAR currents and suppress epileptic activity . Thus, depending on the brain region, DBI can potentiate or inhibit GABAAR activity. Little, however, is 3 known about how DBI exerts these actions. How do products from a single protein elicit positive versus negative effects on GABAAR currents? Do DBI- derived peptides such as ODN elicit the same functional effects as DBI? Do different cell types make and process distinct DBI peptide(s)? Is DBI modified post-translationally (e.g. phosphorylated, acylated) and can these modifications alter DBI's physiological effects? The proposed experiments will address these fundamental questions and will 1) determine if specific GABAAR subunit combinations mediate the positive and negative actions of DBI and ODN; 2) determine if astrocytes and neurons release different DBI peptides and/or post-translationally process DBI differently, which impact its functional effects and 3) determine if astrocytes and neurons express different DBI transcript variants. Successful completion of these experiments will validate DBI's role as an endogenous BZD and will lay the foundation for understanding how DBI regulates GABA- mediated inhibition in the brain.