Periodic Reporting for period 4 - MetAGEn (Metabolic and Genetic Regulation of Ageing) Reporting period: 2020-02-01 to 2020-07-31 Summary of the context and overall objectives of the project Ageing is a complex physiological process that affects almost all species, including humans. Despite its importance for all of us, the biology of ageing is insufficiently understood. To uncover the molecular underpinnings of ageing, I am performing an interdisciplinary research program that will identify and investigate metabolic and genetic regulators of ageing. Progressive loss of cellular homeostasis causes ageing and an age-associated decline in protein quality control has been implicated in numerous diseases, including neurodegeneration. This link between ageing and disease demonstrates the importance of basic research into the biology of ageing. On the one hand, this research offers new insights into fundamental biological processes. On the other hand, a deep understanding of biological underpinnings of the ageing process will open up new ways to address underlying causes for many diseases that affect Western societies. Seeking for ways to improve protein quality, I have identified a novel longevity pathway in Caenorhabditis elegans. In a forward genetic screen, I found a link between metabolites in the hexosamine pathway and cellular protein quality control. Hexosamine pathway activation extends C. elegans lifespan, suggesting modulation of ageing by endogenous molecules.In a first step, I am exploring the mechanism by which hexosamine metabolites improve protein quality control in mammals, using cultured mammalian cells and a mouse model for neurodegeneration. Preliminary data show that hexosamine pathway metabolites enhance proteolytic capacity in cells and reduce protein aggregation, suggesting conservation. Second, I am investigating molecular mechanisms that activate the hexosamine pathway to promote protein homeostasis and counter ageing. Third, I am performing a direct forward genetic screen for modulators of ageing in C. elegans. For the first time, mutagenesis and next generation sequencing can be paired in forward genetic screens to interrogate the whole genome for lifespan-extending mutations in a truly unbiased manner. This innovative approach has the potential to reveal novel modulators of the ageing process.Taken together, this work aims to understand molecular mechanisms that maintain cellular homeostasis to slow the ageing process, and to develop a new technology to identify yet unknown genetic modulators of ageing.Conclusion of the Action:1. I have investigated how the hexosamine pathway modulates cellular protein quality control in mammalian cells. Our published work (Horn et al., 2020 iScience) shows that hexosamine pathway activation is a trigger for a conserved stress response pathway, the integrated stress response (ISR). A mild ISR activation that we observed both in C. elegans and in mammalian cells, activated autophagy that was required to clear toxic protein aggregates. 2. Through biochemical and structural biology approaches we have delineated the molecular regulatory mechanism of GFAT-1 the key enzyme of the hexosamine pathway (Ruegenberg et al., 2020 Nature Commun).3. We expanded, beyond the initial scope of the project, our research to genetic screens in haploid cells which enable high-resolution mechanistical insights in to protein function (Horn et al., 2018 Oncotarget; Allmeroth et al., 2020 Leukemia). This work has also revealed new insights into the regulation of the hexosamine pathway. 4. We performed a forward longevity screen in C. elegans, as outlined in the initial proposal, and have now identified many new longevity loci, one of which revealed a new function of the ISR in longevity (Derisbourg et al., 2020 bioRxiv) Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far I. Role of mammalian GFAT-1 in protein quality control. Using cell biological and biochemical assays, I am investigating if the hexosamine pathway can regulate protein homeostasis in mammals. Within the first reporting period we could show that indeed the toxic aggregation of human disease-associated proteins is reduced upon HP upregulation. This is dependent upon endogenous degradative processes. I will also use a mouse model of neurodegeneration to test if GFAT-1 activation might ameliorate an age-associated disease. We have generated the mouse model and are currently crossing triple-transgenic mice in order to investigate the role of neuron-specific GFAT-1 activation in a mouse model for a neurodegenerative disease. We show that hexosamine pathway activation mildly triggered the ISR and downstream autophagy, that was required for proteoprotection.II. Molecular characterization of GFAT-1. I will investigate the biochemical mechanism of GFAT-1 gof through enzyme kinetic assays and an unbiased search for interaction partners. We have now successfully purified recombinant human GFAT-1 and have established a number of activity assays. This work has revealed the mechanism for the GFAT-1 gain-of-function. We have completed this work and found multiple independent gain-of-function mechanisms of GFAT-1 that are all conserved in the human protein. Loss of feedback inhibition is a key gain-of-function mechanism. III. Unbiased direct forward genetic screen for longevity genes in C. elegans. I will perform the first forward genetic screen that uses longevity as a primary readout. We have now generated more than 100 long-lived C. elegans mutants and have completed next-generation sequencing for all of these within reporting period 1. Analysis of this rich and unique data set has revealed a number of candidates for novel longevity genes are we are in the process of confirming these using the CRISPR/Cas9 technology. Next, we will prioritize on the most interesting mutants and will devise a publication strategy. We have selected a group of new longevity mutants that control protein biosynthesis and the ISR and, through this work, we identified a novel aspect in the regulation of selective protein biosynthesis that directly affects aging. In a project that goes beyond the original proposal, we have adapted haploid mouse embryonic stem cells for mutagenesis screens and implemented an approach that enables screens at amino acid resolution, providing direct insights into protein function. We have applied this in my team as well as in numerous collaborations and have founded a start-up company based on this technology and with help from an ERC PoC grant (ACUSLABS). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) I. GFAT-1 activation in mammalian cells activates the integrated stress response, needed for the cyto-protective effect. II. GFAT-1 activation occurs through loss of feedback inhibition. III. Unbiased direct forward genetic screens for longevity genes in C. elegans have indicated an unexpected role of the integrated stress response in longevity. Crystal Structure of Human GFAT-1
