SNAP-25 EXPRESSION AND HYPERACTIVITY IN COLOBOMA MICE

COLOBOMA 小鼠中的 SNAP-25 表达和多动症

• 批准号: 6126171
• 负责人: MICHAEL COLIN WILSON
• 金额: $ 25.92万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6126171
• 关键词: acetylcholine; attention deficit disorder; behavior test; behavioral /social science research tag; behavioral genetics; body physical activity; developmental neurobiology; disease /disorder model; gene expression; gene targeting; genetic strain; genetically modified animals; laboratory mouse; laboratory rabbit; model design /development; nerve /myelin protein; neural transmission; neurotransmitter transport; protein isoforms; protein structure function; synapses; synaptic vesicles; synaptosomes; syntaxin; tissue /cell culture; transfection

The regulated expression of presynaptic nerve terminal proteins is critical both in developing the circuitry and in controlling neurotransmitter signaling that underlies nervous system function and ultimately behavior. The long-term goal is to define the molecular mechanisms governing the regulation and interactions between these synaptic proteins, and how this contributes to the diversity and plasticity of normal synaptic transmission, and how dysregulation of these processes leads to neurophysiological deficits. Specifically, this investigation addresses the function of SNAP-25, a protein that plays a key role in vesicle docking and regulated exocytosis of neurotransmitters. The mouse mutant, coloboma, bearing a contiguous gene defect and deficient in SNAP-25 expression, implicates dysregulation of SNAP-25 in spontaneous hyperactivity, and with delayed neurobehavorial development, deficits in learning and memory, and at the cellular level in abnormal hippocampal physiology and neurotransmitter release. Mutations affecting SNAP-25 expression may serve as effective models of hyperkinesis, a prominent component of Attention Deficit Hyperactivity Disorder, Tourette syndrome and other neurophysiological disorders. The proposed studies will test the hypothesis that SNAP-25 is involved in these abnormalities, and that two developmentally regulated isoforms of the protein have specialized roles that contribute differently to neural development and mature physiology of synaptic transmission. Towards this goal an integrated approach incorporating the following Specific Aims is proposed: 1) to determine if the phenotypic effects ascribed to the coloboma mutation are specific to SNAP-25. These studies will use Snap gene "rescued" and homologous recombinant null mutants to characterize deficits in neurobehavioral development and learning, in hippocampal electrophysiology, including long-term potentiation and theta EEG activity, and in transmitter release using in vitro synaptosomal preparations. 2) to determine the molecular specificity of SNAP-25 isoforms. Experiments using yeast expression systems and in vitro protein binding assays will characterize the differential interactions of SNAP- 25a and b isoforms with syntaxin and other presynaptic proteins involved in regulated vesicular exocytosis. 3) to define the specific role of the SNAP-25b isoform in neurotransmission. SNAP-25b will be over-expressed in neural cell lines deficient in this isoform, and the cells will be assayed for synaptic vesicle cycling and acetylcholine release as indexes of SNAP-25b function in synaptic transmission. 4) to establish the function of SNAP-25b in the intact nervous system, a homologous recombination strategy will be used to generate mutant mice that are limited to SNAP-25a isoform expression. Analysis of these partial loss- of-function mutants at behaviorial, electrophysiological and neurochemical levels will determine if deficits in mature neurophysiology and behavior result from deficiencies in specialized functions of SNAP- 25b. Through these studies, a better understanding of the molecular processes of neurotransmission will be achieved and, importantly, novel well-defined animal models will be established for the design and characterization of therapeutics targeted to hyperactivity in human neuropsychiatric disorders.

突触前神经末梢蛋白的调节表达是 在开发电路和控制方面都至关重要 神经递质信号传导是神经系统功能的基础 最终的行为。长期目标是定义分子 管理这些因素之间的调节和相互作用的机制 突触蛋白，以及它如何促进多样性和 正常突触传递的可塑性，以及如何失调 这些过程会导致神经生理缺陷。具体来说，这 研究解决了 SNAP-25 的功能，SNAP-25 是一种蛋白质，具有 在囊泡对接和调节胞吐作用中发挥关键作用 神经递质。小鼠突变体，缺损，携带连续基因 SNAP-25 表达的缺陷和缺陷，意味着 SNAP-25 的失调 SNAP-25 导致自发性多动和迟发性神经行为 发育、学习和记忆缺陷以及细胞水平 海马生理学和神经递质释放异常。 影响 SNAP-25 表达的突变可能作为有效的模型 运动过度，注意力缺陷多动的一个重要组成部分 紊乱、抽动秽语综合症和其他神经生理障碍。这 拟议的研究将检验 SNAP-25 参与的假设 这些异常，以及两种发育调节亚型 该蛋白质具有特殊的作用，对神经系统有不同的贡献 突触传递的发育和成熟的生理学。朝这个方向 目标 结合以下具体目标的综合方法是 建议：1）确定表型效应是否归因于 缺损突变是 SNAP-25 特有的。这些研究将使用 Snap 基因“拯救”和同源重组无效突变体来表征 海马神经行为发育和学习缺陷 电生理学，包括长时程增强和 theta 脑电图 活性，以及​​使用体外突触体释放递质 准备工作。 2) 确定SNAP-25的分子特异性 同工型。使用酵母表达系统和体外蛋白质的实验 结合测定将表征 SNAP-的不同相互作用 25a 和 b 亚型，涉及突触融合蛋白和其他突触前蛋白 在受调节的囊泡胞吐作用中。 3）明确具体角色 神经传递中的 SNAP-25b 亚型。 SNAP-25b 将过度表达 在缺乏这种亚型的神经细胞系中，这些细胞将 以突触小泡循环和乙酰胆碱释放为指标进行测定 SNAP-25b 在突触传递中的功能。 4）建立 SNAP-25b 在完整神经系统中的功能，同源 重组策略将用于产生突变小鼠 仅限于 SNAP-25a 同工型表达。分析这些部分损失—— 行为、电生理和功能丧失的突变体 神经化学水平将决定成熟神经生理学是否存在缺陷 和行为是由于 SNAP 专门功能的缺陷造成的 25b.通过这些研究，可以更好地了解分子 神经传递过程将被实现，重要的是，新颖的 将为设计和建立明确的动物模型 针对人类多动症的治疗方法的表征 神经精神疾病。