Targeting novel AMPK effectors in the regulation of healthy aging

描述（由申请人提供）：饮食限制（DR）可以延长健康寿命，预防多种年龄性疾病，包括癌症、神经退行性疾病和糖尿病。不幸的是，DR还会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激活的蛋白激酶（AMPK）是一种细胞内能量传感器，当能量水平较低时被激活，AMPK的激活在哺乳动物和线虫中都体现了DR的作用。一个具有组成性活性的AMPK等位基因（CA-AMPK）可以延长秀丽隐杆线虫的寿命，但与DR一样，它也会导致生育能力下降和形性低下。最近，maair实验室发现保守的creb调控的转录共激活因子（CRTC）是ca - ampk介导的秀丽隐杆线虫长寿的关键下游效应因子。激活的AMPK使秀丽隐杆线虫的CRTC磷酸化，导致核排斥，从而影响其调节转录的能力。一个突变的CRTC等位基因，通过阻断AMPK的磷酸化而呈现组成核，完全抑制CA-AMPK延长寿命，但不阻断CA-AMPK对生育或形态的影响，从而使AMPK长寿信号从不良副作用中分离出来。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调节转录，以响应营养信号，但CRTC在长寿中的作用是完全新的和未被探索的。因此，本研究的中心目标是验证CRTC在ampk调控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和保守联系的假设。通过在特定组织类型中表达CA-AMPK和构成核CRTC等位基因的转基因线虫菌株的工程设计，将揭示AMPK和CRTC在寿命调控中的空间要求。此外，越来越多的证据表明，crtc介导的转录和AMPK依赖的长寿治疗分别需要哺乳动物和秀丽隐杆线虫内质网应激反应的效应物。因此，产生核心内质网应激介质和AMPK和/或CRTC组成活性等位基因基因缺失的转基因动物，将使我们能够验证内质网应激信号作为线虫AMPK-CRTC长寿的保守机制的一部分的假设。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存测试。最后，RNA-seq分析将确定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的培训中受益匪浅，包括RNA-seq和哺乳动物细胞培养。

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.