Development of Tethered Toxins for Neuroscience Research

描述(由申请人提供)： 这项资助的目的是产生一系列新的药物，用于体内受体、离子通道和信号分子的遗传操作。这些药物(栓系毒素)是通过GPI锚定或跨膜将自然产生的多肽神经毒素拴在细胞表面上而产生的嵌合分子。这些研究源于哺乳动物原毒素基因(例如Lynx 1)的发现，这些基因是蛇毒毒素的进化先驱，可以作为天然GPI锚定形式的nAChRs的调节器。初步结果是，拴系的银环蛇毒素在nAChRs上保持其活性，并且它们不会从细胞表面剥离以抑制相邻细胞。数以千计的自然产生的多肽神经毒素(如银环蛇毒素、芋螺毒素、刀豆素等)的存在，它们精致的靶标特异性，以及利用BAC转基因小鼠在体内靶向表达这些毒剂的能力，表明开发一种通用的策略来利用它们的效力在体内使用将允许对广泛的神经元功能进行遗传控制。例如，细胞对神经活动、神经递质受体功能(如ACh、NMDA、5-HT3受体)的特定遗传控制，以及特定的GPCR信号转导级联将成为可能。其具体目的是：1)构建更多的拴系毒素，特别是拴系毒素，并测试它们在非洲爪哇卵母细胞中的活性；2)在体内获得表达特定CNS细胞类型的拴系毒素的BAC转基因小鼠。通过评估基于特定目标1的结果和靶向受体和离子通道在体内的作用的当前知识所预期的表型来评估系留毒素的有效性；3)开发可诱导的系留毒素，以提高这一策略的时间分辨率，用于特定细胞和信号通路的遗传操作。这些研究将在体内对中枢神经系统发育、功能和功能障碍所需的功能进行前所未有的基因解剖。

DESCRIPTION (provided by applicant): The purpose of this grant is to generate a novel series of agents for genetic manipulation of receptors, ion channel, and signaling molecules in vivo. These agents (tethered toxins) are chimeric molecules derived from tethering of naturally occurring peptide neurotoxins to the cell surface via GPI anchors or transmembrane. These studies derive from the discovery of mammalian prototoxin genes (e.g. Lynx 1) which are the evolutionary antecedents of snake venom toxins, and which can function as modulators of nAChRs in their native GPI-anchored form. Preliminary results are that tethered bungarotoxins retain their activity on nAChRs, and that they are not cleaved from the cell surface to inhibit adjacent cells. The existence of many thousands of naturally occurring peptide neurotoxins (e.g. bungarotoxins, conotoxins, conantokins, etc.), their exquisite target specificities, and the ability to target expression of the agents in vivo using BAC transgenic mice, suggests that the development of a generic strategy for harnessing their potency for in vivo use will permit genetic control over a wide variety of neuronal functions. For example, cell specific genetic control of neural activity, neurotransmitter receptor function (e.g. ACh, NMDA, 5-HT3 receptors), and specific GPCR signal transduction cascades would become possible. The specific aims are to: 1) Construct additional tethered toxins, particularly tethered conotoxins, and test their activity in Xenopus oocytes; 2) Produce BAC transgenic mice expressing tethered toxins in specific CNS cell types in vivo. Assess the efficacy of tethered toxin action by evaluating phenotypes that would be expected based on results obtained in Specific Aim 1 and current knowledge of the roles of the targeted receptors and ion channels in vivo; 3) Develop inducible tethered toxins to improve the temporal resolution of this strategy for genetic manipulation of specific cells and signaling pathways. These studies will allow unprecedented precision in the genetic dissection of functions required for CNS development, function and dysfunction in vivo.