Mechanisms of Ras signaling in single synapses

描述（由申请人提供）：小GTPase蛋白Ras对突触连接的调节至关重要，如突触传递的加强，新突触的形成和细胞兴奋性的调节。Ras对于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信号的失败与导致认知障碍和学习缺陷的疾病有关，如自闭症、x连锁智力迟钝和神经纤维瘤病1。尽管Ras信号在突触可塑性中的重要性已得到充分认识，但目前尚不清楚Ras如何解码和传递钙动力学以调节其多种下游效应。在神经元中，Ras信号参与跨越不同隔室的信号事件，包括脊髓、树突和细胞核。因此，Ras信号的时空动态在确定其下游效应方面可能是重要的。为了研究Ras在神经元中的信号传导机制，我们最近开发了一种荧光技术，使我们能够在脑组织深处的活神经元中以单突触分辨率成像Ras的活性，利用这种技术，本提议的目的是了解Ras信号传导的时空调节机制。我们的假设是，Ras信号的时空模式是由以下因素决定的：1)涉及多种激酶和反馈回路的钙依赖性信号网络控制的Ras激活；2)由于Ras和Ras调节因子的扩散和运输，Ras激活的空间扩散。为了验证这一假设，我们将利用双光子谷氨酸释放技术，对单个脊柱上谷氨酸受体激活后，脊髓和树突中的Ras活性进行成像。我们的初步数据表明，Ras激活发生在受刺激的脊柱，随后扩散到其母树突和附近的脊柱。本研究的具体目标是：1)识别激活单个脊柱中Ras的上游信号；2)确定Ras在树突中的空间调节机制和作用；3)阐明Ras GTPase家族差异激活的机制。这项工作将促进我们对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.