A 'Mitochondria-nucleus-ribosome' Signaling Axis Controls Lifespan in Mit Mutants

描述(申请人提供)：这项研究的长期目标是改善美国老年人的健康和福祉。我们的直接目标是研究线粒体功能的中断是如何与衰老过程相联系的。近年来，人们已经清楚地看到，线粒体电子传输链的缺陷与许多与年龄相关的疾病有关，包括心脏病、II型糖尿病、帕金森病、阿尔茨海默氏症和癌症。线粒体是细胞中产生能量的关键部位。15%的美国人口患有这些慢性退行性疾病。虽然还不能说线粒体导致了这些问题，但很明显，线粒体的变化参与其中，因为它们的功能发生了可测量的变化。也许最令人惊讶的是，在包括老鼠、苍蝇、线虫和酵母在内的几个物种中发现了一些突变体，它们对线粒体电子传递链功能障碍的反应自相矛盾，寿命延长。对这一耐人寻味的突变家族的研究为线粒体及其在衰老中的作用提供了新的线索。 在这项研究中，我们将研究秀丽线虫MIT突变体的衰老，这些突变体都是长寿的，含有缺陷的线粒体电子传输链活性。我们发现了这些突变体中一种新颖的三组分信号级联反应的证据，它将线粒体ETC活动的破坏与核检查点信号、细胞质核糖体翻译的阻断和寿命控制联系起来。这是一个完全出乎意料的发现，因为这些过程跨越了三个不同的细胞隔间。我们目前的研究将确定这种新的信号级联的原因和影响。我们假设，MIT突变体中核苷酸合成的中断是其核检查点反应激活的基础。我们已经确定了在这些动物中激活的三种检查点蛋白，现在我们的初步数据指向一个直接的核糖体靶点，该靶点可能通过调节核编码蛋白向线粒体的运输而成为关键的长寿控制点。我们的具体目标将被确定是否在麻省理工学院突变体中改变了核糖核苷酸的生物合成。我们还将确定这些动物中是否存在核转录停滞。最后，我们将测试我们识别的核糖体靶标在检查点激活后是否确实差异地控制核编码的线粒体等转录本的翻译。我们将通过使用强大的生化和遗传工具来解决这些问题。我们将使用LC-MS/MS来量化核苷酸池的变化，定量PCR来确定转录停滞是否发生，并结合转基因报告、多聚体图谱和RNA序列来确定核编码的线粒体蛋白是否在MIT突变体中被差异地阻止翻译。

DESCRIPTION (provided by applicant): The long-term goal of this study is to improve the health and well-being of older Americans. Our immediate goal is to examine how disruption of mitochondrial function is linked with the aging process. In recent years, it has become clear that defects in the electron transport chain of mitochondria, the key site of energy production in cells are linked with many age-related diseases - including heart disease, Type II diabetes, Parkinson Disease, Alzheimer's dementia, and cancer. 15% of the US population suffers from these chronic degenerative disorders. While it cannot yet be said that mitochondria cause these problems, it is clear that changes in mitochondria are involved, because their function is measurably altered. Perhaps most surprising has been the discovery of a number of mutants, across several species including mice, flies, nematodes and yeast, that paradoxically respond to mitochondrial electron transport chain dysfunction with increases in their lifespan. Investigation of this intriguing family of mutants is shedding new light on mitochondria and their role in aging. In this study, we will examine aging in the Caenorhabditis elegans Mit mutants, which are all long-lived and contain defective mitochondrial electron transport chain activity. We have uncovered evidence for a novel, three component signaling cascade in these mutants that links disruption of mitochondrial ETC activity with nuclear checkpoint signaling, blockade of cytoplasmic ribosomal translation, and lifespan control. This is a completely unexpected finding because these processes span three distinct cellular compartments. Our current study will define the cause and effects of this novel signaling cascade. We hypothesize that disruption of nucleotide synthesis in Mit mutants underlies activation of their nuclear checkpoint response. We have identified three checkpoint proteins that are activated in these animals, and now our preliminary data points toward a direct ribosomal target that potentially acts as a key longevity control point by regulating trafficking of nuclear-encoded proteins to the mitochondria. Our specific objectives will be determined if the biosynthesis of ribonucleotides is altered in Mit mutants. We will also determine if there is nuclear transcriptional stalling in these animals. Finally we will test whether our identified ribosomal target does indeed differentially act to control translation of nuclear-encoded mitochondrial ETC transcripts following checkpoint activation. We will address these questions through the use of powerful biochemical and genetic tools. We will use LC-MS/MS to quantify changes in nucleotide pools, quantitative PCR to determine if transcriptional stalling is occurring, and a combination of transgenic reporters, polysomal profiling and RNA Seq to determine if nuclear-encoded mitochondrial proteins are differentially prevented from being translated in Mit mutants.