Final Report Summary - ER LONGEVITY (Improving Protein Homeostasis to Extend Health- and Lifespan) The career integration grant has essentially supported my career over the past 4 years. It has provided substantial support to my postdoctoral training and in the transition to becoming an independent principal investigator in 2014. In the course of my work as a postdoctoral researcher I tested the hypothesis that protein homeostasis in the endoplasmic reticulum (ER) significantly promotes cellular stress resistance that, at an organism level, can result in extended health and life span. To address this point, I used the genetic model organism Caenorhabditis elegans in forward genetic screens. I identified a novel longevity pathway that utilizes endogenous amino sugar metabolites to improve protein homeostasis and extend lifespan in the nematode. This work was published in the journal Cell in 2014. Difference between work expected and carried outBelow is a list of the original milestones for the sponsorship period with a discussion of completed work as well as an explanation of initially expected work and work carried out:Aim I Identification of systemic regulators of ER stress pathways: I first focused on the broad unbiased screen of Aim II, and identified a metabolite, UDP-GlcNAc as a mediator of proteotoxic stress resistance and longevity. Given these results, we decided not to carry out the experiments detailed in Aim I but rather to focus on a potential role of GlcNAc as a systemic regulator of ER homeostasis. Addition of GlcNAc to the medium extends lifespan, which shows that it can act cell-non-autonomously. This is indeed a conserved mechanism. In cultured neuronal cells, GlcNAc supplementation likewise improves protein quality control and suppresses the formation of toxic protein aggregates. We have now initiated experiments with mice in which we are testing the effect of GlcNAc supplementation in a model of proteotoxic stress. Characterization of systemic regulation of ER-UPR during heat stress:We assayed heat stress resistance in various C. elegans mutants, including the GFAT-1 gain-of-function mutants and found that TM resistance and longevity are not necessarily linked to heat stress resistance. Aim II Identification of novel genes that extend C. elegans lifespan through improving ER homeostasis:We identified point mutations in the C. elegans gfat-1 gene that result in gain-of-function. The resulting activation of the HP extends C. elegans lifespan. In-depth analysis of regulatory pathways linking ER-UPR to lifespan in C. elegans:We have mechanistically described the molecular changes downstream of GFAT-1 gain-of-function. We show that distinct quality control mechanisms, including ERAD and autophagy, were induced. Interestingly, this improves global protein quality control as toxicity from cytosolic protein aggregates was alleviated as well. This work of Aim II has been published in the journal Cell in 2014Aim III Test relevance of C. elegans longevity genes in tissue culture assays:In my independent laboratory, we have made significant progress in this regard. We use the CRISPR/Cas9 technology to introduce GFAT-1 gain-of-function mutations in the respective mouse gene, which results in gain-of-function. The resultant UDP-GlcNAc elevation in cells likewise promotes resistance to proteotoxic stress. Next, we will investigate the upstream mechanisms that link UDP-GlcNAc levels with improvements of protein quality control in this system. Biochemical characterization of candidate genes in tissue culture:One remaining major milestone for the second funding period was to understand at biochemical detail how the 4 independent amino acid substations identified in our screen induce GFAT-1 activity. To achieve this, have successfully expressed soluble and active GFAT-1 from insect cell cultures. Using this system we can now compare the various mutants regarding their enzymatic activity. One possible mechanism for GFAT-1 gain-of-function is the loss of UDP-GlcNAc mediated feedback inhibition.
