Somatic Ca2+ Signaling in CNS Neurons

描述(由申请人提供)： 神经元活动产生细胞内钙离子的动态变化，这些钙离子在包括基因表达在内的许多细胞功能的调节中发挥核心作用。最近，我们证明了早期发育的小脑浦肯野神经元表达一种膜到核的体细胞钙信号通路，该通路是在树突状细胞表达之前的发育重要阶段由内源性电活动驱动的。我们的初步结果表明，该信号通路的激活产生胞浆和核内钙信号，这些信号导致转录因子的激活和下游细胞蛋白水平的变化。我们假设，在未成熟的Purkinje神经元中驱动这一途径的内源性活动是一个与发育相关的信号，该活动的一个作用是诱导启动下一个发育阶段所必需的基因表达的变化，即功能树突的表达。作为检验这一假说的第一步，我们将对在相关发育阶段受到各种刺激范例的急性分离的浦肯野神经元进行基因阵列微观分析。这种方法将使我们能够同时分析神经元激活对大量已知对神经元功能和发育有贡献的神经元转录本的影响。通过使用这些信息，结合使用电生理和钙成像技术的蛋白质表达和功能分析，我们将能够了解钙调控基因表达在特定发育阶段的影响。我们还将确定膜到核的体细胞钙信号通路的分子组成和功能特性，并确定基因芯片微分析确定的蛋白质基因表达所必需的条件。这些研究的结果将与浦肯野神经元的发育计划直接相关，浦肯野神经元的发育计划在形态水平上已经得到了很好的表征，并涉及到不同的和可识别的发育阶段。然而，这些研究确定的一般原理也将有助于我们理解活动依赖的基因表达在其他类型中枢神经系统神经元发育过程中的作用。

DESCRIPTION (provided by applicant): Neuronal activity produces dynamic changes in cellular calcium that play a central role in the regulation of a number of cellular functions including gene expression. Recently we demonstrated that early developing cerebellar Purkinje neurons express a membrane-to-nucleus somatic calcium signaling pathway that is driven by endogenously generated electrical activity at a developmentally significant stage, just prior to dendritic expression. Our preliminary results show that activation of this signaling pathway produces cytosolic and nuclear calcium signals and that these signals result in activation of transcription factors and changes in the level of downstream cellular proteins. We hypothesize that the endogenously generated activity that drives this pathway in the immature Purkinje neurons is a developmentally relevant signal and that one role for the activity is to induce changes in gene expression necessary for initiation of the next developmental stage, expression of functional dendrites. As a first step toward testing this hypothesis, we will apply gene array microanalysis to acutely isolated Purkinje neurons subjected to various stimulation paradigms at the relevant developmental stage. This approach will enable us to simultaneously analyze the effects of neuronal activation on a large number of neuronal transcripts known to contribute to neuronal function and development. By using this information in combination with analysis of protein expression and functional analysis using electrophysiological and calcium imaging techniques we will be able to gain an understanding of the impact of calcium-regulated gene expression at a specific developmental stage. We will also identify the molecular components and functional properties of the membrane-to-nucleus somatic calcium signaling pathway and determine the conditions that are essential for gene expression of proteins identified by the gene array microanalysis. Results from these studies will be directly relevant to the developmental program of Purkinje neurons, which has been well characterized on a morphological level and involves distinct and identifiable developmental stages. However, the general principles identified by the studies will also contribute significantly to our understanding of the role of activity-dependent gene expression during development in other CNS neuronal types.