SNAP-25 EXPRESSION AND HYPERACTIVITY IN COLOBOMA MICE

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很关键 神经递质信号是神经系统功能和 最终的行为。长期的目标是定义分子 监管机制以及它们之间的相互作用 突触蛋白，以及这如何有助于多样性和 正常突触传递的可塑性，以及 这些过程会导致神经生理缺陷。具体地说，这 研究涉及SNAP-25的功能，SNAP-25是一种发挥 在囊泡对接和调节胞吐中的关键作用 神经递质。突变的小鼠，Coloboma，携带一个连续基因 SNAP-25表达缺陷和缺陷，提示细胞功能失调 Snap-25在自发性多动和神经行为延迟中的作用 发育、学习和记忆缺陷，以及细胞水平 在异常的海马区生理和神经递质释放。 影响SNAP-25表达的突变可作为有效的模型 多动症--注意缺陷多动的一个重要组成部分 精神障碍、多发性抽动症和其他神经生理学障碍。这个 拟议的研究将检验SNAP-25参与的假设 这些异常，以及发育调节的两种亚型 这种蛋白质具有特殊的作用，对神经的贡献不同 突触传递的发展和成熟生理学。朝向这个方向 目标结合了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 错配突变是SNAP-25所特有的。这些研究将使用Snap 基因“被拯救”和同源重组零突变体的特征 海马区神经行为发育和学习的缺陷 电生理，包括长时程增强和theta脑电 活性，以及利用体外突触体释出递质 准备工作。2)确定SNAP-25的分子特异性 异构体。利用酵母表达系统和体外蛋白的实验 结合分析将表征SNAP-的差异相互作用。 25a和b与突触素和其他突触前蛋白有关的亚型 在调节的囊泡胞吐作用中。3)确定 神经传递中的Snap-25b亚型。Snap-25b将过度表达 在缺乏这种异构体的神经细胞系中，细胞将是 以突触小泡循环和乙酰胆碱释放为指标 研究SNAP-25b在突触传递中的作用。4)建立 SNAP-25b在完整神经系统中的作用 重组策略将用于产生突变小鼠，这些小鼠 仅限于SNAP-25a亚型表达。对这些部分损失的分析- 在行为、电生理和 神经化学水平将决定成熟神经生理学的缺陷 而行为是由于SNAP的专业功能不足-- 25B。通过这些研究，更好地理解了分子 将实现神经传递的过程，重要的是，新的 将为设计和设计建立定义良好的动物模型 针对人类多动症的治疗药物的特征 神经精神障碍。

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.