Targeting novel AMPK effectors in the regulation of healthy aging

描述（由申请人提供）：饮食限制（DR）可延长健康寿命，并预防各种年龄发病性疾病，包括癌症、神经退行性疾病和糖尿病。不幸的是，DR也带来了不必要的心理和生理副作用，使其成为不切实际的治疗方案。AMP活化蛋白激酶（AMPK）是一种细胞内的能量传感器，当能量水平较低时被激活，AMPK的激活再现了哺乳动物和C.优雅一个组成性激活的AMPK等位基因（CA-AMPK）可提高C. elegans，但像DR一样，也会导致生育力降低和亚型。最近，Mair实验室鉴定了保守的CREB调节的转录辅激活因子（CRTC）作为CA-AMPK介导的C.优雅活化的AMPK磷酸化C.线虫CRTC，引起核排斥，从而影响其调节转录的能力。通过阻断AMPK磷酸化使突变CRTC等位基因组成性核化，完全抑制CA-AMPK延长寿命，但不阻断CA-AMPK对生育力或形态学的影响，从而使AMPK长寿信号与不期望的副作用解偶联。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颖且未探索的。因此，该建议的中心目标是检验CRTC作为AMPK调节的衰老中营养、能量稳态和体细胞维持之间的关键和保守联系的假设。转基因C.仅在特定组织类型中表达CA-AMPK和组成型核CRTC等位基因的秀丽线虫菌株将揭示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C.优雅因此，产生具有核心ER应激介质和AMPK和/或CRTC的组成型活性等位基因的组合遗传缺失的转基因动物将允许我们测试ER应激信号传导作为C中AMPK-CRTC长寿的保守机制的一部分的假设。优雅我们在C.然后将测试秀丽线虫在哺乳动物细胞中的保存情况。最后，RNA-seq分析将确定AMPK-CRTC信号传导如何调节基因表达以促进长寿。总之，这些研究将阐明一种新的和治疗上可行的机制，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大大受益于拟议技术和模型的培训，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.