Deciphering Molecular Mechanisms of Calcium Homeostasis

项目总结/摘要 钙是许多细胞过程的关键介质，包括肌肉收缩， 线粒体活性、转录、细胞分裂和突触囊泡释放。巧合的是， 钙还可引发细胞死亡和细胞坏死。因此，钙的失调 信号传导可以破坏细胞内稳态。因此，钙水平需要严格 控制。事实上，钙信号传导缺陷与许多神经退行性疾病有关。 疾病、肌肉萎缩症和心脏病。为了了解调节 钙信号传导以及这个过程中的缺陷如何导致细胞功能障碍，我们正在 利用模型系统秀丽隐杆线虫来识别关键过程， 参与钙处理的监管。我们最近已经证明，SEL-12， C. elegans早老素直系同源物，在介导内质网-线粒体 钙稳态早老素功能的破坏导致线粒体内 钙水平和改变线粒体代谢促进蛋白质稳态崩溃， 神经变性早老素是从植物到人类发现的高度保守的蛋白质， 广泛存在于内膜结构上（例如，内质网和溶酶体） 大多数细胞类型。然而，早老素在内膜系统中的作用尚不清楚。 重要的是，人类早老素的突变是早发性家族性遗传性痴呆最常见的原因。 老年痴呆症尽管早老素参与阿尔茨海默氏症的鉴定， 早老素突变的功能性后果， 老年痴呆症是不了解的。为了进一步了解早老素在 线粒体钙稳态和神经元的健康，我们已经开发了一种新的和高度 选择性RNA干扰筛选，以确定基因产物，冥想提高 在SEL-12突变体中观察到内质网到线粒体钙信号传导。从这个 筛选，我们已经确定了几个蛋白质已知介导内质网钙 释放和线粒体钙摄取，但我们也发现了几种基因产物， 在内质网和线粒体钙信号传导中的未表征的作用。我们提出 利用多方面的方法，结合基因操作，高分辨率活细胞， 显微镜下分析内质网和线粒体动力学，线粒体 活性测定和行为测定，以确定这些基因产物以及自噬的作用， 12在线粒体健康和神经元健康。

PROJECT SUMMARY/ABSTRACT 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 disrupt cellular homeostasis. 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.