Targeting novel AMPK effectors in the regulation of healthy aging

Burkewitz, Kristopher

Abstract

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.

Public Health Relevance

Patient age is a critical risk factor common to a number of seemingly diverse pathologies, including cancer, neurodegenerative disease, cardiovascular disease, stroke, and type II diabetes. Dietary restriction and the genetic pathways that sense and regulate nutrient levels can improve healthy aging, however, and studies in animal models have shown that targeting these pathways provides broad therapeutic benefits against many age-onset diseases. The proposed studies will reveal novel therapeutic targets against age-related pathologies by elucidating the molecular-level mechanism linking nutrient-sensing to pro-longevity gene expression.