Molecular Genetics of Caloric Restriction in Aging Flies

描述(申请人提供)：卡路里限制(CR)是延长动物寿命和延缓与年龄相关的症状发生的最可靠的方法。在目前的资助期间，我们在了解年龄和卡路里摄入量对成年果蝇生理和寿命的影响方面做出了重大贡献。果蝇Indy基因的突变极大地延长了寿命。Indy是一种Krebs循环中间产物的二羧酸转运体，主要存在于对中间代谢重要的组织中。印地活动减少的延长寿命的效应被认为是一种遗传CR，这一假说得到了在这一资助期间进行的生化、分子和遗传学研究的支持。对印地长寿果蝇基因组转录反应的测定揭示了在新陈代谢中起作用的基因的下调--特别值得注意的是线粒体氧化磷酸化(OP)复合体I和III的成分表达一过性下降。我们表明，在Indy果蝇中，OP I和III复合体具有较低的酶活性，产生较少的活性氧物种(ROS)，导致较低的氧化损伤。然而，Indy Fly的ATP产量与对照组相似，这一结果可以用Indy Fly中发现的线粒体密度增加来解释。考虑到线粒体在能量产生和细胞内稳态中的关键作用，我们的初步数据在代谢和长寿途径之间提供了额外的联系，并形成了我们的假设的基础，即OP复合体的瞬时变化介导了Indy突变果蝇的长寿。同时，我们已经证明了rpd3组蛋白脱乙酰酶的下调，或基因上dSir2组蛋白脱乙酰酶的过度表达，或通过喂食白藜芦醇提高dSir2的活性，通过类似于CR的机制延长了果蝇的寿命。这给了我们一个机会来确定线粒体生理学上的类似变化是否是三种遗传CR模型寿命延长的潜在途径的一部分。在这项建议的目标1中，我们将确定Indy、rpd3和Sir2基因或CR的遗传操作是否通过下调每种寿命延长条件下OP I和III组分的水平和活性来影响寿命。在目标2中，我们将确定降低复合体I和III的成分水平是否会对苍蝇的生理和寿命产生影响。在目标3中，我们将研究OP I和III组分之间的遗传交互作用以及已建立的Indy/rpd3/Sir2长寿途径。在目标4中，我们将通过评估线粒体在Indy/rpd3/Sir2长寿途径中的生理学和生物发生来进一步阐明线粒体在CR寿命延长中的作用。由于线粒体在能量平衡、应激反应和寿命方面的作用是众所周知的，我们提议的实验将把现有的知识扩展到OP成分在CR途径中的新作用，并可能为治疗干预提供基础。与公共健康相关：在脊椎动物和无脊椎动物物种中，限制卡路里已成为保护有机体免受衰老有害影响的最有效方法。本项目将研究果蝇通过热量限制延长寿命的分子机制。它将揭示减少卡路里摄入量如何影响新陈代谢和寿命，并为开发治疗人类年龄相关疾病的新疗法提供基础。

DESCRIPTION (provided by applicant): Caloric restriction (CR) is the surest way of increasing life span and delaying the onset of age-related symptoms in animals. During the current funding period, we made significant contributions to our understanding of the effects of age and caloric intake on physiology and longevity of adult Drosophila. Mutation in the Indy gene in the fruit fly, Drosophila melanogaster, dramatically extends life span. INDY is a dicarboxylate transporter of Krebs cycle intermediates primarily found in the tissues important for intermediary metabolism. The life extending effect of reduced Indy activity has been proposed to result in a form of genetic CR, a hypothesis supported by biochemical, molecular and genetic studies carried out during this funding period. Determination of the genomic transcriptional responses of Indy long-lived flies reveal down-regulation of genes that function in metabolism-particularly noteworthy is a transient decrease in the expression of components of the mitochondrial oxidative phosphorylation (OP) complexes I and III. We showed that in Indy flies OP I and III complex have lower enzyme activity, produced less reactive oxygen species (ROS), and caused lower oxidative damage. However, production of ATP in Indy flies is similar to the control, a result that could be explained by increased mitochondrial density found in Indy flies. Considering the crucial role of mitochondria in energy production and cellular homeostasis, our preliminary data provide additional links between metabolic and longevity pathways and form the basis for our hypothesis that transient change in the OP complexes mediate longevity in Indy mutant flies. In parallel, we have shown that down-regulation of the rpd3 histone deacetylase, or overexpression of dSir2 histone deacetylase genetically, or increasing dSir2 activity by feeding flies resveratrol, extends life span in Drosophila by a mechanism similar to CR. This give us an opportunity to determine if similar changes in mitochondrial physiology are part of the pathway underlying life span extension in three fly models of genetic CR. In aim 1 of this proposal, we will determine if genetic manipulations of Indy, rpd3, and Sir2 genes, or CR, effect longevity by downregulation of the levels and activity of the OP I and III components in each life span extending condition. In aim 2, we will determine if decreasing the levels of components of complex I and III have effects on fly physiology and longevity. In aim 3, we will examine genetic interactions between OP I and III components and the established Indy/rpd3/Sir2 longevity pathway. In aim 4, we will further elucidate the role mitochondria play in CR life span extension by assessing the mitochondrial physiology and biogenesis in the Indy/rpd3/Sir2 longevity pathway. Since the role of mitochondria in energy homeostasis, stress response and longevity is well known, our proposed experiments will extend current knowledge to the novel role of the OP components in the CR pathway and potentially provide a basis for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Caloric restriction has emerged as the most efficient way to protect the organism against deleterious effects of aging in both vertebrate and invertebrate species. This project will study the molecular mechanism underlying life span extension in fruit flies, Drosophila melanogaster, by caloric restriction. It will reveal how reduced caloric intake affects metabolism and life span, and provide the foundation for the development of new therapies for the treatments of age-associated diseases in humans.