Demystifying Disorders of Bicistronic Calcium Channel Genes

摘要 这个项目的长期目标是解释复杂基因型-表型的基础 越来越多的严重钙通道基因疾病的关系，并开发基于 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， 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， 和一种新发现的转录因子我们已经发现转录因子是由 通过类似于内部核糖体进入位点（IRES）的过程进行内部翻译。我们假设 这些Ca 2+通道基因的突变可能具有多种结果，在不同的情况下， 神经元放电、钙信号传导、转录因子表达的调节或直接改变神经元的功能。 转录因子的功能。本研究将系统地探讨其功能和生物学特性， 这三种新的转录因子的作用，它们的表达是如何被IRES调节的， 细胞生理学控制着它们从细胞核到细胞核的易位，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/或存活力受损， 这些不同的疾病。我们将使用在原代神经元中表达的重组钙通道来研究这一点 以及来自正常和患者来源的人类重编程神经元，以及在转基因小鼠中表达良好的 特征突变。我们将使用下一代方法研究基因结合和表达， 使用表位和荧光标签与生理刺激的核转位，使用双重免疫刺激的IRES功能， 荧光素酶报告基因和免疫印迹，使用免疫荧光显微镜的神经元发育， 使用表达转录因子的反义寡核苷酸、miRNA和AAV病毒载体的矫正疗法。

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.