MOLECULAR MECHANISMS MEDIATING NEURONAL PLASTICITY.

具体目标 对神经元可塑性调节分子机制的理解取得了进展， 为药物滥用和成瘾的病理生理学提供了重要线索。虽然数量可观， 在阐明这一过程的几个关键方面取得了进展，例如NMDA的作用， AMPA受体分别介导活动诱导的可塑性的诱导和表达， 这一过程的关键特征仅得到部分理解。例如，现在很明显， 突触可塑性的形式需要已经预先定位在突触中的mRNA的局部翻译。 突触附近。然而，我们才刚刚开始了解 介导mRNA运输和翻译。此外，最近的研究将注意力集中在 与突触可塑性相关的形态学变化的普遍性和重要性。然而，在这方面， 这些变化是如何发生的，以及它们与功能可塑性的关系还很不清楚。因此，委员会认为， 人们对了解介导和调节这些的分子机制有着浓厚的兴趣， 流程. 为了深入了解突触可塑性的这些关键特征，我们计划专注于两个项目。 一方面，我们将研究Tech的功能和调节，Tech是一种富含大脑的RhoA GEF， 与调节树突形态有关。在另一个，我们将研究的作用， 翻译蛋白/Trax RNA结合复合物介导树突中的RNA运输 Tech：神经元RhoA GEF 最近的研究表明，Rho家族的小GTP酶控制多功能的 在调节神经元形态中起关键作用的细胞内信号通路网络 和突触可塑性。这种信号通路在突触可塑性中的重要性已经被证实。 Rho信号通路的异常会导致智力迟钝，这一发现强调了这一点。 此外，最近的一些研究也证实了它与药物滥用的相关性，这些研究将药物滥用的变化与药物滥用之间的关系联系起来， LIM激酶及其底物cofilin的活性，Rho信号通路的两个下游效应物， 可卡因的长期影响。为了帮助确定Rho信号通路的调节和作用， 神经元，我们已经开始表征RhoA GEF，称为Tech，它在大脑中高度富集。在 初步研究，我们发现在培养的神经元中过表达Tech结构， 对树突形态的显著影响，并且还降低了表面GluR 1染色的水平。在 旨在了解技术如何监管的研究，我们发现，与其他RhoGEF家族一样， 成员，它包含一个自抑制域。此外，我们最近的研究结果表明， 在该结构域内的一个位点经历酪氨酸磷酸化。因此，我们计划： I.检查Tech缺失对神经元形态和AMPA受体的影响 贩运和 二.检验Tech GEF活性受酪氨酸磷酸化调节的假设。 翻译蛋白/Trax RNA结合复合物 在以前的研究中，我们已经确定了翻译蛋白和Trax作为RNA结合的亚基， 该复合物与介导树突状RNA运输有关。令人信服的证据 支持这一假设的是最近与E. Tongiorgi。简而言之，我们发现BDNF mRNA与翻译蛋白/Trax复合物共沉淀， 以及Translin siRNA抑制海马培养物中BDNF mRNA的树突运输。作为 这些发现提供了“原理证明”，即翻译蛋白/Trax复合物介导树突状细胞 RNA的运输，我们计划： I.识别与大脑中的翻译蛋白/Trax复合物结合的其他转录物， 二.评估翻译蛋白/Trax复合物在调节这些蛋白定位中的作用。 成绩单

SPECIFIC AIMS Advances in understanding the molecular mechanisms mediating neuronal plasticity have provided important clues to the pathophysiology of drug abuse and addiction. Although substantial progress has been made in elucidating several key aspects of this process, e.g. the role of NMDA and AMPA receptors in mediating the induction and expression of activity-induced plasticity, respectively, critical features of this process are only partially understood. For example, it is now clear that enduring forms of synaptic plasticity require local translation of mRNAs that have been pre-positioned in the vicinity of synapses. However, we are only beginning to understand the mechanisms and molecules mediating mRNA trafficking and translation. In addition, recent studies have focused attention on the prevalence and importance of morphological changes associated with synaptic plasticity. However, how these changes occur and their relevance to functional plasticity are poorly defined. Accordingly, there is intense interest in understanding the molecular machinery that mediates and regulates these processes. To gain insights into these key features of synaptic plasticity, we plan to focus on two projects. In one, we will investigate the function and regulation of Tech, a RhoA GEF enriched in brain that has been implicated in regulating dendritic morphology. In the other, we will examine the role of the Translin/Trax RNA binding complex in mediating RNA trafficking in dendrites.. Tech: a neuronal RhoA GEF Recent studies have demonstrated that the Rho family of small GTPases controls a versatile network of intracellular signaling pathways that play a pivotal role in regulating neuronal morphology and synaptic plasticity. The importance of this signaling pathway in synaptic plasticity has been underscored by the finding that abnormalities in Rho signaling pathways lead to mental retardation. Furthermore, its relevance to drug abuse has been supported by recent studies linking changes in the activity of LIM kinase and its substrate cofilin, two downstream effectors of the Rho signaling pathway, to long-term effects of cocaine. To help define the regulation and role of the Rho signaling pathway in neurons, we have begun to characterize a RhoA GEF, called Tech, that is highly enriched in brain. In preliminary studies, we have found that overexpression of Tech constructs in cultured neurons triggers prominent effects on dendritic morphology and also reduces the level of surface GluR1 staining. In studies aimed at understanding how Tech is regulated, we have found that, like other RhoGEF family members, it contains an autoinhibitory domain. Furthermore, our recent findings suggest that Tech undergoes tyrosine phosphorylation at a site within this domain. Accordingly, we plan to: I. Examine the impact of Tech deletion on neuronal morphology and AMPA receptor trafficking, and II. Test the hypothesis that Tech GEF activity is regulated by tyrosine phosphorylation. Translin/Trax RNA binding complex In previous studies, we have identified Translin and Trax as subunits of an RNA binding complex that has been implicated in mediating dendritic RNA trafficking. Compelling evidence supporting this hypothesis has emerged from recent studies conducted in collaboration with Dr. E. Tongiorgi. In brief, we have found that BDNF mRNA co-precipitates with the Translin/Trax complex and that Translin siRNA suppresses dendritic trafficking of BDNF mRNA in hippocampal cultures. As these findings have provided "proof of principle" that the Translin/Trax complex mediates dendritic trafficking of RNA, we plan to: I. Identify other transcripts that bind to the Translin/Trax complex in brain, and II. Assess the role of the Translin/Trax complex in regulating the localization of these transcripts.