Targeting novel AMPK effectors in the regulation of healthy aging

靶向新型 AMPK 效应物调节健康衰老

• 批准号: 8527019
• 负责人: Kristopher Burkewitz
• 金额: $ 4.92万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8527019
• 关键词: 5' -AMP-activated protein kinase; Adverse effects; Age; Age of Onset; Aging; Alleles; Animal Model; CREB1 gene; Caenorhabditis elegans; Cardiovascular Diseases; Cell Culture Techniques; Chronic; Communication; Cues; Diabetes Mellitus; Disease; Eating; Engineering; Exclusion; Experimental Genetics; Family; Fertility; Foundations; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Goals; Health Benefit; Histocompatibility Testing; Homeostasis; Human; Intervention; Intestines; Link; Liver; Longevity; Longevity Pathway; Maintenance; Malignant Neoplasms; Malnutrition; Mammalian Cell; Mammals; Mediating; Mediator of activation protein; Metabolic; Metabolism; Modeling; Molecular; Morphology; Muscle; Nature; Nematoda; Neurodegenerative Disorders; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nutrient; Nutritional; Output; Pathology; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Physiological; Play; Process; RNA Interference; Regulation; Research; Risk Factors; Role; Scientist; Signal Transduction; Stress; Stroke; System; Techniques; Testing; Therapeutic; Tissues; Training; Transcription Coactivator; Transcriptional Regulation; Transgenic Animals; Transgenic Organisms; age related; biological adaptation to stress; cell type; detection of nutrient; dietary restriction; healthy aging; improved; longevity gene; mutant; new therapeutic target; novel; novel therapeutic intervention; nutrition; prevent; programs; psychologic; public health relevance; research study; response; sensor; skills; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): Dietary restriction (DR) extends healthy lifespan and protects against a wide array of age-onset diseases, including cancer, neurodegenerative disease, and diabetes. Unfortunately, DR also carries unwanted psychological and physiological side effects that make it an impractical therapeutic regime. AMP-activated protein kinase (AMPK) is an intracellular energy sensor activated when energy levels are low, and activation of AMPK recapitulates the effects of DR in both mammals and C. elegans. A constitutively active AMPK allele (CA-AMPK) enhances longevity in C. elegans, but like DR, also causes reduced fertility and hypomorphism. Recently, the Mair lab identified the conserved CREB-regulated transcriptional coactivator (CRTC) as the critical downstream effector of CA-AMPK-mediated longevity in C. elegans. Activated AMPK phosphorylates C. elegans CRTC, causing nuclear exclusion and thus impacting its ability to regulate transcription. A mutant CRTC allele, rendered constitutively nuclear by blocking AMPK phosphorylation, completely suppresses lifespan extension by CA-AMPK, but does not block CA-AMPK effects on fertility or morphology, thus uncoupling AMPK longevity signaling from undesirable side effects. Previous studies in mammalian models have demonstrated that CRTC regulates transcription through interactions with conserved bZip-family transcription factors in response to nutrient signals, but the role of CRTC in longevity is completely novel and unexplored. Thus, the central goal of this proposal is to test the hypothesis that CRTC serves as a critical and conserved link between nutrition, energy homeostasis, and somatic maintenance in AMPK-regulated aging. Engineering of transgenic C. elegans strains expressing CA-AMPK and constitutively nuclear CRTC alleles only in specific tissue types will reveal the spatial requirements for AMPK and CRTC in lifespan regulation. Additionally, mounting evidence suggests that CRTC-mediated transcription and AMPK- dependent longevity treatments independently require effectors of the ER stress response in mammals and C. elegans. Therefore, generating transgenic animals with combined genetic deletions of core ER stress mediators and constitutively active alleles of AMPK and/or CRTC will allow us to test the hypothesis that ER stress signaling functions as part of a conserved mechanism in AMPK-CRTC longevity in C. elegans. Our findings in C. elegans will then be tested for conservation in mammalian cells. Lastly, RNA-seq analysis will define how AMPK-CRTC signaling regulates gene expression to promote longevity. Taken together these studies will elucidate a novel and therapeutically amenable mechanism linking energy and metabolic signals to a gene expression program promoting healthy aging. Further, my capabilities and potential as an independent scientist will benefit greatly from training in the proposed techniques and models, including RNA-seq and mammalian cell culture.

描述（由申请人提供）：饮食限制 (DR) 可延长健康寿命并预防多种老年性疾病，包括癌症、神经退行性疾病和糖尿病。不幸的是，DR 还带来不必要的心理和生理副作用，使其成为一种不切实际的治疗方案。 AMP 激活蛋白激酶 (AMPK) 是一种细胞内能量传感器，在能量水平较低时激活，AMPK 的激活重现了 DR 对哺乳动物和秀丽隐杆线虫的影响。组成型活跃的 AMPK 等位基因 (CA-AMPK) 可以延长线虫的寿命，但与 DR 一样，也会导致生育力下降和低等态。最近，Mair 实验室发现保守的 CREB ​​调节转录共激活因子 (CRTC) 是线虫中 CA-AMPK 介导的长寿的关键下游效应子。激活的 AMPK 会使秀丽隐杆线虫 CRTC 磷酸化，导致核排斥，从而影响其调节转录的能力。突变的 CRTC 等位基因通过阻断 AMPK 磷酸化而构成核，完全抑制 CA-AMPK 延长寿命，但不会阻断 CA-AMPK 对生育力或形态的影响，从而使 AMPK 长寿信号与不良副作用脱钩。先前在哺乳动物模型中的研究表明，CRTC 通过与保守的 bZip 家族转录因子相互作用来响应营养信号来调节转录，但 CRTC 在长寿中的作用是完全新颖且未经探索的。因此，该提案的中心目标是检验这样的假设：CRTC 在 AMPK 调节的衰老过程中作为营养、能量稳态和体细胞维持之间的关键且保守的联系。仅在特定组织类型中表达 CA-AMPK 和组成型核 CRTC 等位基因的转基因线虫菌株的工程将揭示 AMPK 和 CRTC 在寿命调节中的空间要求。此外，越来越多的证据表明，CRTC 介导的转录和 AMPK 依赖性长寿治疗独立地需要哺乳动物和线虫中 ER 应激反应的效应器。因此，产生具有核心 ER 应激介质和 AMPK 和/或 CRTC 组成型活性等位基因的组合遗传删除的转基因动物将使我们能够检验这样的假设：ER 应激信号传导作为 C.线虫。我们在秀丽隐杆线虫中的发现随后将在哺乳动物细胞中进行保守性测试。最后，RNA-seq 分析将定义 AMPK-CRTC 信号如何调节基因表达以促进长寿。总而言之，这些研究将阐明一种新颖的、治疗上可行的机制，将能量和代谢信号与促进健康衰老的基因表达程序联系起来。此外，我作为一名独立科学家的能力和潜力将从所提出的技术和模型（包括 RNA-seq 和哺乳动物细胞培养）的培训中受益匪浅。