Demystifying Disorders of Bicistronic Calcium Channel Genes

摘要 这个项目的长期目标是解释复杂的基因-表型的基础。 与越来越多的严重钙通道基因紊乱的关系和开发基于 这些新的见解。为了取得这些进展，我们将利用我们的新发现，至少有三个 这些钙通道基因是双顺反子的，即它们编码两种不同的蛋白质，即钙通道蛋白 和一种新发现的转录因子。我们发现转录因子是由 由类似于内部核糖体进入位点(IRES)的过程进行的内部翻译。我们假设 这些钙离子通道基因的突变可能会产生不同的结果，在不同的情况下， 神经元放电，钙信号，转录因子表达的调节或直接改变 转录因子的功能。在本研究中，我们将系统地探索其功能和生物学特性。 这三个新转录因子的作用，它们的表达是如何由IRES调控的，如何正常 细胞生理学支配它们移位到细胞核和从细胞核移位，以及不同的突变如何影响 通道门控、IRES功能和转录因子导致神经元发育和/或存活率受损 这些不同的紊乱。我们将使用在初级神经元中表达的重组钙通道来研究这一点。 来自正常来源和患者来源的人类重新编程的神经元，以及在转基因小鼠中表达良好的 以突变为特征。我们将使用下一代方法研究基因结合和表达， 利用表位和荧光标记结合生理刺激的核转位，IRES使用DUAL 荧光素酶报告和免疫印迹，神经元发育使用免疫荧光显微镜和 使用反义寡核苷酸、miRNA和表达转录因子的AAV病毒载体进行纠正治疗。

Abstract The long term objectives of this project are to explain the basis for the complex genotype-phenotype relationships for a growing number of severe calcium channel gene disorders and develop therapies based on these new insights. To make these advances we will capitalize on our new discovery that at least three of these calcium channel genes are bicistronic i.e. they encode two distinct proteins, the calcium channel proteins and a newly discovered transcription factor. We have discovered that the transcription factor is translated by internal translation by a process resembling an internal ribosomal entry site (IRES). We hypothesize that mutations in these Ca2+ channel genes may have a diversity of outcomes, affecting, in distinct cases, neuronal firing, calcium signaling, regulation of the expression of the transcription factor or directly altering the function of the transcription factor. In this study we will systematically explore the function and biological action of these three novel transcription factors, how their expression is regulated by the IRES, how normal cellular physiology governs their translocation to and from the nucleus, and how different mutations affecting channel gating, IRES function and transcription factor cause impaired neuronal development and or viability in these different disorders. We will study this using recombinant calcium channels expressed in primary neurons and human reprogrammed neurons from normal and patient sources, and in transgenic mice expressing well characterized mutations. We will study gene binding and expression using next generation approaches, nuclear translocation using epitope and fluorescent tags with physiological stimuli, IRES function using dual luciferase reporters and immunoblotting, neuronal development using immunofluorescent microscopy and corrective therapy using antisense oligos, miRNA and AAV viral vectors expressing transcription factors.