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）可以延长健康的寿命，并预防各种年龄发作的疾病，包括癌症，神经退行性疾病和糖尿病。不幸的是，DR还具有不必要的心理和生理副作用，使其成为不切实际的治疗状况。 AMP激活的蛋白激酶（AMPK）是一种当能量水平较低时激活的细胞内能传感器，AMPK的激活概括了DR在哺乳动物和秀丽隐杆线虫中的影响。组成型活性AMPK等位基因（CA-AMPK）可以增强秀丽隐杆线虫的寿命，但与DR一样，也会导致生育能力和不形成性降低。最近，MAIR实验室确定了保守的CREB调节的转录共激活因子（CRTC）是秀丽隐杆线虫中Ca-Ampk介导的寿命的关键下游效应器。活化的AMPK磷酸化秀丽隐杆线虫CRTC，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磷酸化来使组成性核能完全抑制CA-AMPK的寿命延长，但不会阻止CA-AMPK对生育能力或形态的影响，从而使AMPK longevity longevity longevity longevity longevity信号从不良副作用中产生。先前在哺乳动物模型中的研究表明，CRTC通过与保守的BZIP家庭转录因子相互作用来调节转录，以响应营养信号，但是CRTC在寿命中的作用是完全新颖的，尚未开发。因此，该提案的核心目标是检验以下假设：CRTC是营养，能量稳态和AMPK调节衰老中的躯体维持之间的关键和保守联系。仅在特定组织类型中表达CA-AMPK和组成性核CRTC等位基因的转基因秀丽隐杆线虫菌株的工程将揭示在寿命调节中对AMPK和CRTC的空间要求。此外，越来越多的证据表明，CRTC介导的转录和依赖性寿命治疗独立地要求哺乳动物和秀丽隐杆线虫中ER应力反应的效应子。因此，生成具有核心ER应力介体和AMPK和/或CRTC组成型活性等位基因的遗传缺失的转基因动物将使我们能够检验以下假说，即ER应力信号在AMPK-CRTC longevity中的保守机制的一部分是秀丽杆菌中的AMPK-CRTC寿命。然后，我们在秀丽隐杆线虫中的发现将在哺乳动物细胞中进行保护。最后，RNA-seq分析将定义AMPK-CRTC信号如何调节基因表达以促进寿命。综上所述，这些研究将阐明一种新型且可及时的机制，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，我作为独立科学家的能力和潜力将从提出的技术和模型（包括RNA-Seq和哺乳动物细胞培养）中的培训中受益匪浅。