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 神经系统功能的神经递质信号传导， 最终行为。长期目标是确定分子 管理这些调节和相互作用的机制 突触蛋白，以及这如何有助于多样性， 正常突触传递的可塑性，以及 这些过程导致神经生理缺陷。具体来说， 研究解决了SNAP-25的功能，SNAP-25是一种蛋白质， 在囊泡对接和调节胞吐中的关键作用 神经传递素携带连续基因的小鼠突变体coloboma SNAP-25表达的缺陷和不足，暗示着 SNAP-25在自发性多动和迟发性神经营养不良中的作用 发育，学习和记忆缺陷，以及在细胞水平 海马体生理和神经递质释放异常。 影响SNAP-25表达的突变可以作为有效的模型， 运动机能亢进是注意缺陷多动障碍的一个重要组成部分 抽动秽语综合征和其他神经生理障碍。的 拟议的研究将测试SNAP-25参与的假设， 这些异常，以及两种发育调节的亚型， 这些蛋白质具有特殊作用， 突触传递的发育和成熟生理学。为实现这一 目标是一种综合方法，包括以下具体目标， 建议：1）确定是否归因于 缺失突变对SNAP-25是特异性的。这些研究将使用Snap 基因“拯救”和同源重组无效突变体，以表征 神经行为发育和学习缺陷，海马 电生理学，包括长时程增强和θ EEG 活性，并在递质释放使用体外突触体 准备工作2)确定SNAP-25的分子特异性 同种型。使用酵母表达系统和体外蛋白质的实验 结合分析将表征SNAP的差异相互作用， 25 a和B同种型与突触融合蛋白和其他突触前蛋白有关 调节囊泡胞吐作用。3)明确了政府的具体作用， SNAP-25 b亚型在神经传递中的作用SNAP-25 b将被过度表达 在缺乏这种同种型的神经细胞系中，细胞将 测定突触囊泡循环和乙酰胆碱释放作为指标 SNAP-25 b在突触传递中的功能。4)建立 SNAP-25 b在完整神经系统中的功能， 重组策略将用于产生突变小鼠， 仅限于SNAP-25 a同种型表达。分析这些部分损失- 在行为，电生理和 神经化学水平将决定是否在成熟的神经生理缺陷 和行为是由于SNAP的专门功能的缺陷造成的， 25 b.通过这些研究，更好地了解分子 神经传递的过程将被实现，重要的是， 将为设计建立明确的动物模型， 针对人类多动症的治疗剂的表征 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.