Deciphering Molecular Mechanisms of Calcium Homeostasis

项目总结 钙是许多细胞过程的关键媒介，包括肌肉收缩， 线粒体活性、转录、细胞分裂和突触小泡释放。矛盾的是， 钙还会引发细胞死亡和细胞坏死。因此，钙的调节失调 信号可以以不同的方式和不同的程度影响细胞。因此，钙的水平 需要严格控制。事实上，钙信号的缺陷在许多方面都有牵连。 神经退行性疾病、肌肉营养不良和心脏病。要了解 调节钙信号的机制以及这一过程中的缺陷如何导致细胞 功能障碍，我们正在利用模型系统秀丽线虫来识别关键的 参与钙处理调节的过程。我们最近做了 证明线虫早老素直系同源基因SEL-12具有中介作用 内质网-线粒体钙动态平衡。早老素功能紊乱 导致线粒体钙水平升高并改变线粒体代谢 促进蛋白质稳态崩溃和神经退行性变。早老素是一种高度的 从植物到人类的保守蛋白，广泛存在于内膜上 大多数细胞类型的结构(如内质网和溶酶体)。然而，这个角色 早老素在内膜系统中的作用尚不清楚。重要的是，人类的突变 早老素是早发性家族性阿尔茨海默病最常见的原因。尽管 20多年前早老素与阿尔茨海默病的关系 导致阿尔茨海默病的早老素突变的功能后果不是 明白了。为了进一步了解早老素在线粒体钙中的作用 动态平衡和神经元适合性，我们已经开发出一种新型的、高选择性的RNA 干扰筛选鉴定内质网升高的基因产物 在sel-12突变体中观察到的线粒体钙信号转导。在这个屏幕上，我们有 确定了几种已知的介导内质网钙释放和 线粒体钙摄取，但我们也鉴定了几个基因产物与一个 在内质网和线粒体钙信号中的作用尚未确定。我们建议 利用结合了基因操作、高分辨率活细胞的多方面方法 显微镜下分析内质网和线粒体动力学，线粒体 活性分析和行为分析，以确定这些基因产物以及选择的作用。 12例具有线粒体健康和神经元健康。

PROJECT SUMMARY Calcium is a critical mediator of many cellular processes, including muscle contraction, mitochondrial activity, transcription, cell division, and synaptic vesicle release. Paradoxically, calcium can also trigger cell death and cellular necrosis. Therefore, dysregulation in calcium signaling can affect cells in different ways and to varying degrees. Consequently, calcium levels need to be tightly controlled. Indeed, defective calcium signaling has been implicated in many neurodegenerative diseases, muscular dystrophies and heart disease. To understand the mechanisms regulating calcium signaling and how defects in this process can lead to cellular dysfunction, we are exploiting the model system Caenorhabditis elegans to identify critical processes that are involved in the regulation of calcium handling. We have recently demonstrated that SEL-12, the C. elegans presenilin ortholog, has a role in mediating endoplasmic reticulum-mitochondrial calcium homeostasis. Disruption of presenilin function results in an increase in mitochondrial calcium levels and alters mitochondrial metabolism promoting protein homeostasis collapse and neurodegeneration. Presenilin is a highly conserved protein found from plants to humans that is extensively found on endomembrane structures (e.g., endoplasmic reticulum and lysosome) of most cell types. However, the role presenilin has in the endomembrane system is not clear. Importantly, mutations in human presenilin are the most common cause of early onset familial Alzheimer's disease. Despite the identification of the involvement of presenilin in Alzheimer's disease over 20 years ago, the functional consequences of mutations in presenilin causing Alzheimer's disease are not understood. To gain further insight into the role presenilin has in mitochondrial calcium homeostasis and neuronal fitness, we have developed a novel and highly selective RNA interference screen to identify gene products that meditate the elevated endoplasmic reticulum to mitochondrial calcium signaling observed in sel-12 mutants. From this screen, we have identified several proteins known to mediate endoplasmic reticulum calcium release and mitochondrial calcium uptake but we also identified several gene products with an uncharacterized role in endoplasmic reticulum and mitochondrial calcium signaling. We propose to utilize a multifaceted approach that combines genetic manipulation, high resolution live cell microscopy to analyze endoplasmic reticulum and mitochondrial dynamics, mitochondrial activity assays, and behavioral assays to determine the role these gene products as well as sel- 12 have in mitochondrial health and neuronal fitness.