PROJECT SUMMARY Dietary restriction (DR) is the most potent method for promoting healthy aging and age-onset disease resistance in animal models. However, DR’s therapeutic potential is limited by associated negative physiological effects, including impaired growth, immunity and reproductive capacity. Although nutrientsensing mediators of DR have been identified, such as mTOR, FOXO/As and the sirtuins, these central nodes recapitulate the entirety of the response, making them sub-optimal therapeutic targets. Our long-term objective is to uncover molecular mechanisms that specifically mediate only the pro-longevity effects of DR to develop optimal therapeutics. A key mediator of DR is AMP-activated protein kinase (AMPK), a cellular fuel gauge activated when energy levels are low. However, like DR, AMPK increases lifespan at the cost of impaired growth and reproduction. The objective in this application is to use the genetically tractable model system C. elegans to identify mechanisms by which AMPK and DR specifically mediates longevity, in order to elucidate the first molecular targets that recapitulate only the pro-health effects of DR. The central hypothesis is that beneficial and detrimental effects of DR can be uncoupled. In support of this hypothesis, specific amino acid combinations in the diet have recently been shown to increase lifespan while maintaining normal reproduction, establishing that the positive effects of DR on lifespan do not require obligate detrimental side effects. In addition, in the previous funding period we demonstrated that the transcriptional coactivator and AMPK direct target CRTC-1 could uncouple the longevity effects of AMPK from other negative side effects such as developmental delay, reproduction suppression and reduced body size. We now seek to identify the mechanisms by which CRTC-1 specifically mediates longevity. The rationale for this project is that, before we can generate viable therapies from DR for clinical application we must first identify mechanisms that 1) recapitulate only the positive effects of DR and 2) are effective when applied late in life, post-diagnosis of age-related disease. Based upon strong preliminary data we will test three specific aims. 1) We will examine the functional role of neuronal CRTC-1 in promoting healthy aging. 2) We will use CRISPR editing of endogenous AMPK targets to delineate the roles of additional pathways downstream of this key energy sensor 3) We will define the contribution of mitochondrial and peroxisome remodeling in AMPK and DR longevity. Collectively, we expect this work to provide the first example of molecular pathways that uncouple the positive and negative effects of DR, a critical step in transitioning DR research to the promotion of healthy human aging.