Molecular Genetics of Caloric Restriction in Aging Flies

描述（由申请人提供）：热量限制（CR）是延长动物寿命和延缓年龄相关症状发作的最可靠方法。在目前的资助期内，我们在理解年龄和热量摄入对成年果蝇生理和寿命的影响方面做出了重大贡献。果蝇（Drosophila melanogaster）体内Indy基因的突变可以显著延长寿命。INDY是克雷布斯循环中间体的二羧酸转运体，主要存在于对中间体代谢重要的组织中。Indy活性降低的寿命延长效应已被提出导致某种形式的遗传CR，这一假设得到了本资助期间进行的生化、分子和遗传学研究的支持。对印度长寿果蝇基因组转录反应的测定揭示了代谢功能基因的下调——特别值得注意的是线粒体氧化磷酸化（OP）复合物I和III组分表达的短暂减少。我们发现，在Indy蝇中，OP I和OP III复合物具有较低的酶活性，产生较少的活性氧（ROS），并造成较低的氧化损伤。然而，Indy果蝇中ATP的产生与对照组相似，这一结果可以用Indy果蝇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稳态中的关键作用，我们的初步数据提供了代谢和长寿途径之间的额外联系，并为我们的假设奠定了基础，即OP复合物的短暂变化介导了Indy突变果蝇的寿命。同时，我们已经证明rpd3组蛋白去乙酰化酶的下调，或dSir2组蛋白去乙酰化酶的过表达，或通过给果蝇喂食白藜芦醇来增加dSir2活性，通过类似于CR的机制延长果蝇的寿命。这使我们有机会确定线粒体生理学的类似变化是否是三种遗传CR模型中果蝇寿命延长的途径的一部分。我们将确定Indy、rpd3和Sir2基因或CR的基因操作是否会通过下调OP I和OP III成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物I和III成分的水平是否对果蝇的生理和寿命有影响。在目标3中，我们将研究OP I和OP III组分与已建立的Indy/rpd3/Sir2长寿途径之间的遗传相互作用。在目标4中，我们将通过评估Indy/rpd3/Sir2长寿通路中的线粒体生理和生物发生，进一步阐明线粒体在CR寿命延长中的作用。由于线粒体在能量稳态、应激反应和长寿中的作用是众所周知的，我们提出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。公共卫生相关性：热量限制已成为保护生物体免受脊椎动物和无脊椎动物衰老有害影响的最有效方法。本项目将研究通过热量限制延长果蝇（Drosophila melanogaster）寿命的分子机制。它将揭示减少热量摄入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.