Mechanisms of Ras signaling in single synapses

描述(由申请人提供)：小的GTPase蛋白RAS对许多神经元过程是重要的，这些过程对于调节突触连接是必不可少的，例如加强突触传递，形成新的突触和调节细胞的兴奋性。RAS对长期维持突触可塑性所需的蛋白质合成和基因转录也很重要。与RAS信号在突触可塑性中的重要作用一致，RAS信号的失败与引起认知障碍和学习障碍的疾病有关，如自闭症、X-连锁智力低下和神经纤维瘤病1。虽然RAS信号在突触可塑性中的重要性得到了很好的认识，但RAS信号如何解码和传递钙动力学来调节其不同的下游效应尚不清楚。在神经元中，RAS信号涉及跨越不同隔室的信号事件，包括棘突、树突和细胞核。因此，RAS信号的时空动态很可能是决定其下游效应的重要因素。为了研究神经元中RAS信号的机制，我们最近发展了一种荧光技术，使我们能够以单一突触分辨率成像活的神经元中的RAS活动，利用这一技术，本提议的目的是了解RAS信号的时空调节机制。我们的假设是，RAS信号的时空模式是由1)受涉及多个激酶和反馈环的钙依赖信号网络控制的RAS激活，以及2)由于RAS和RAS调节因子的扩散和运输而引起的RAS激活的空间扩散。为了验证这一假说，我们将使用双光子谷氨酸去除法来成像棘突和树突中RAS活性，以响应单个脊椎上谷氨酸受体的激活。我们的初步数据表明，RAS的激活发生在受刺激的脊椎，随后扩散到其母树突和附近的脊椎。这一建议的具体目的是：1)确定激活单个脊椎中RAS的上游信号；2)确定树突中RAS空间调控的机制和作用；3)阐明RAS GTP酶家族差异激活的机制。这项工作将增进我们对RAS如何将钙与突触可塑性以及最终与学习和记忆相结合的理解。此外，我们的研究将为RAS相关精神障碍的分子机制提供见解。

DESCRIPTION (provided by applicant): The small GTPase protein Ras is important for many neuronal processes essential to the regulation of synaptic connections such as strengthening of synaptic transmission, formation of new synapses and regulation of cell excitability. Ras is also important for protein synthesis and gene transcription required for long-term maintenance of synaptic plasticity. Consistent with essential roles of Ras signaling in synaptic plasticity, failures in Ras signaling are associated with diseases causing cognitive impairments and learning deficits such as autism, X-linked mental retardation and neurofibromatosis 1. Although the importance of Ras signaling in synaptic plasticity is well recognized, it is not clear how Ras decodes and relays calcium dynamics to regulate its diverse downstream effects. In neurons, Ras signaling is involved in signaling events spanning different compartments, including spines, dendrites and the nucleus. Thus, the spatiotemporal dynamics of Ras signaling are likely to be important in determining its downstream effects. To study Ras signaling mechanism in neurons, we have recently developed a fluorescence technique that allows us to image Ras activity with single synapse resolution in living neurons deep in brain tissue, using this technique, the objective of this proposal is to understand the mechanisms of spatiotemporal regulation of Ras signaling. Our hypothesis is that the spatiotemporal pattern of Ras signaling is shaped by 1) Ras activation controlled by calcium-dependent signaling networks involving multiple kinases and feedback loops, and 2) spatial spreading of Ras activation due to the diffusion and trafficking of Ras and Ras regulators. To test this hypothesis, we will image Ras activity in spines and dendrites in response to activation of glutamate receptors on a single spine using 2-photon glutamate uncaging. Our preliminary data suggested that Ras activation occurs at the stimulated spine, subsequently spreading into its parent dendrite and nearby spines. The specific aims of this proposal are to 1) identify upstream signaling that activates Ras in individual spines, 2) determine the mechanisms and roles of the spatial regulation of Ras in dendrites, and 3) elucidate mechanisms underlying differential activation of the Ras GTPase family. This work will advance our understanding of how Ras couples calcium with synaptic plasticity, and ultimately with learning and memory. Moreover, our study will provide insights into the molecular mechanisms underlying Ras-related mental disorders.