Mechanisms of Ras signaling in single synapses

描述（由申请人提供）：小GT3蛋白Ras对于调节突触连接所必需的许多神经元过程是重要的，例如加强突触传递、形成新突触和调节细胞兴奋性。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信号传导在突触可塑性中的重要作用一致，Ras信号传导的失败与引起认知障碍和学习缺陷的疾病如自闭症、X连锁精神发育迟滞和神经纤维瘤病1相关。尽管Ras信号在突触可塑性中的重要性已被充分认识，但Ras如何解码和传递钙动力学以调节其多样化的下游效应尚不清楚。在神经元中，Ras信号传导涉及跨越不同隔室的信号传导事件，包括棘、树突和核。因此，Ras信号的时空动态可能是重要的，在确定其下游的影响。为了研究Ras信号在神经元中的作用机制，我们最近开发了一种荧光技术，该技术允许我们在脑组织深处的活神经元中以单突触分辨率成像Ras活性，使用该技术，本提案的目的是了解Ras信号的时空调节机制。我们的假设是，Ras信号传导的时空模式是由1）Ras激活控制的钙依赖性信号网络，涉及多个激酶和反馈环，2）空间扩散的Ras激活，由于Ras和Ras调节剂的扩散和运输。为了验证这一假设，我们将图像Ras活动的棘和树突上的谷氨酸受体激活一个单一的棘使用2光子谷氨酸解开。我们的初步数据表明，Ras激活发生在刺激的棘，随后蔓延到其母树突和附近的棘。该提案的具体目标是1）鉴定激活单个棘中Ras的上游信号，2）确定树突中Ras的空间调节的机制和作用，以及3）阐明Ras GTfamily的差异激活的潜在机制。这项工作将推进我们对Ras如何将钙与突触可塑性结合，并最终与学习和记忆结合的理解。此外，我们的研究将提供深入了解Ras相关精神障碍的分子机制。

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.