Universität zu Köln

Becker, Ben (2014). Small Molecule Modulators of Dauer Formation and Longevity in Caenorhabditis Elegans. PhD thesis, Universität zu Köln.

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Abstract

7-ketocholesterol modulates dauer formation and longevity via DAF-12 Ageing is the inevitable fate of most living organisms, and is the greatest risk factor for many diseases such as diabetes, cancer, cardiovascular disorders and neurodegeneration. Deceleration of ageing delays the onset of such diseases and improves health into old age. On the cellular level, several signaling pathways that integrate the nutrient sensors insulin/IGF-1-like signaling (IIS) and target of rapamycin (TOR) play an important role in the modulation of lifespan. Downstream effectors of these pathways include broad cellular functions such as protein translation, mitochondrial activity, autophagy and protein homeostasis, affecting youthfulness of cells and the whole organism. Some of these lifespan modulators were found to be direct targets of small molecules such as glucose and rapamycin. In this study, we asked how might endogenous small molecule metabolites including components of the diet such as sugars, amino acids and fatty acids modulate lifespan? What signaling pathways are involved? How can they be used to alter the onset of age-related diseases and potentially help to develop drugs or even prevent them? Interestingly, many pathways and signals regulating lifespan in C. elegans were originally discovered for their role in regulating the dauer decision. C. elegans arrest at the stress-resistant long-lived dauer stage in response to harsh environmental conditions such as high temperature, food scarcity, or high population density. In brief, inhibition the IIS pathway, and resulting transcriptional activity of phosphorylated DAF-16/FOXO activates a hormone biosynthetic pathway that converts cholesterol into bile acid like steroids called the dafachronic acids (DA). DAs are endogenous ligands of the steroid receptor DAF-12, a homolog of mammalian LXR/FXR/VDR, and key determinant of dauer formation. Liganded DAF-12 promotes reproductive development and normal life. Conversely, when these pathways are down-regulated, the unliganded DAF-12 represses these programs and promotes dauer formation and long life. To identify novel signaling molecules regulating life span, we performed a screen for small molecule metabolites that modulate dauer formation in the insulin receptor mutant daf-2(e1368) background. daf-2(e1368) displays a temperature sensitive constitutive dauer formation (Daf-c) phenotype that can be enhanced or suppressed. Our premise was that molecules identified as dauer modulators may also be good candidates for modulating lifespan. We supplemented small molecules involved in energy homeostasis and metabolism such as sugars, amino acids, fatty acids and steroids, and measured dauer formation at a semi-permissive temperature as first readout. From such screens we identified sugars (glucose, galactose, trehalose), amino acids (tryptophan, glycine) and a fatty acid (arachidonic acid) that reduce dauer formation, suggesting they could activate reproductive programs. Interestingly, we also identified 7-ketocholesterol (7-KC) as potent synergistic enhancer of daf-2 dauer formation and focused on this molecule as a potential modulator of longevity. Although 7-KC had little effect on wild type dauer formation, it enhanced dauer formation of various Daf-c mutants of the dauer signaling pathways including daf-2/InsR, daf-7/TGFβ, the Niemann Pick Type C1 homologs, as well as several mutants involved in DA production such as daf-36/Rieske oxygenase. Using a biochemical GC-MS approach, we found that 7-KC altered sterol profiles and exhibited an increase in whole body cholesterol but a decrease in 7-dehydrocholesterol, suggesting possible effects on the first step in Δ7-DA synthesis. Moreover, 7-KC induced hypodermal daf-9/CYP27A1 expression, a marker of mild DA depletion in wild type animals. Importantly, 7-KC was found to extend median lifespan of wild type animals by 20%, in a manner independent of DAF-16/FOXO but dependent on DAF-12/FXR, as well as on the hormone biosynthetic enzyme DAF-9/CYP27A1. In vivo mRNA analyses revealed that 7-KC modestly interferes with expression of DAF-12 target genes (mir-84, mir-48 and mir-241). Accordingly, competition assays suggest that 7-KC thwarts DAF-12 transcriptional activity in cell culture. Whole transcriptome analyses (RNAseq) in C. elegans revealed that 7-KC induces changes in gene expression consistent with regulatory effects on DAF-12 as well as the DAF-12 related receptor, NHR-8. The inferred changes in gene expression suggests that 7-KC opposes known DAF-12 target gene expression and in addition potentially drives a different subset of genes. Taken together, we hypothesize that 7-KC might be converted to an alternative DA-like molecule, 7-keto-DA by DAF-9. In this view, 7-keto-DA might be an alternate DAF-12 ligand, mediating the observed phenotypes. 7-KC is found in all living organisms including humans, where it may be involved in the regulation of bile acid and de novo cholesterol synthesis. Moreover 7-KC was shown to be involved in the formation of age related atherosclerotic cardiovascular disease. Our findings might lead to identification of novel direct mammalian 7-KC target genes and might provide a first step to clarify if 7-KC plays a causative role in atherosclerosis.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Becker, Ben benbecker81@yahoo.de UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-57002
Date:	28 July 2014
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language C. elegans, 7-ketocholesterol, longevity, dauer formation, small molecules UNSPECIFIED
Date of oral exam:	7 April 2014
Referee:	Name Academic Title Antebi, Adam Prof. Dr. Hoppe, Thorsten Prof. Dr. Paulsson, Mats Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/5700