The roles of ccf-1 and pal-1 genes in aging and stress resistance of Caenorhabditis elegans

The CCR4-NOT complex is a critical regulator of gene expression involved in multiple cellular processes, including transcriptional regulation, RNA metabolism, and stress responses. In this thesis, I investigated the role of ccf-1, a component of the CCR4-NOT complex, in stress responses and its necessity for normal lifespan and its implications in various pro-longevity signaling pathways in C. elegans. Through my experiments using environmental stressors such as cadmium and acrylamide, I measured several stress-responsive gene expression levels and observed that ccf-1 is required for stress-induced transcriptional changes in the model organism Caenorhabditis elegans. Additionally, I found that the ccf-1 gene plays an important role in the lifespan of several long-lived mutants, highlighting its significance in the aging process. I also showed that other components of the CCR4-NOT complex are also involved in promoting proper transcriptional response to environmental stressors. Furthermore, my research led to the discovery of the PAL-1 protein as a novel transcriptional factor involved in stress response with pleiotropic effects, demonstrating additive impacts when interacting with the ccf-1 gene. These findings contribute to a better understanding of the molecular mechanisms governing stress responses and aging in C. elegans and may have broader implications in unraveling similar processes in higher organisms, including humans

Longitudinal Studies Of Caenorhabditis Elegans Aging And Behavior Using A Microfabricated Multi-Well Device

The roundworm C. elegans is a powerful model organism for dissecting the genetics of behavior and aging. The central genetic pathways regulating lifespan, such as insulin signaling, were first identified in worms. C. elegans is also the only animal for which a full map of all neural synpatic connections, or connectome, exists. However, current manual and automated methods are unable to efficiently monitor and quantify behavioral phenotypes which unfold over long time scales. Therefore, it has been difficult to study phenotypes such as long-term behavior states and behavioral changes with age in worms. To address these limitations, here I describe a novel device, called the WorMotel, to longitudinally monitor behavior in up to 240 single C. elegans on time scales encompassing the worm\u27s maximum lifespan of two months. The WorMotel is fabricated from polydimethylsiloxane from a 3-D printed negative mold. Each device consists of 240 individual wells, each of which houses a single worm atop agar and bacterial food. I use custom software to quantify movement between frames to longitudinally monitor behavior for each animal. I first describe the application of the WorMotel to the automation of lifespan measurements in C. elegans, the characterization of intra-strain and inter-strain variability in behavioral decline, the relationship between behavior and lifespan, and the scaling of behavioral decline with increasing stress. I then describe the application of the WorMotel to quantify locomotive behavioral states and their modulation by the presence or absence of food as well as biogenic amine neurotransmitters. Using the WorMotel in combination with genetics and pharmacology, I outline a neural circuit by which the biogenic amines serotonin and octopamine regulate locomotion state to signal animals to adopt behavior appropriate to a fed and fasting state, respectively. I include protocols for construction of custom imaging rigs and requirements for long-term imaging as an appendix. The WorMotel is a powerful tool that can facilitate discovery and understanding of the mechanisms underlying long-term phenotypes such as behavioral states and aging

Neuronal SKN-1B Modulates Nutritional Signalling Pathways and Mitochondrial Networks to Control Satiety

Correct responses to nutrient type and availability are a crucial mater for all living organisms. In animals, food-related decisions require the communication of the sensory nervous system with internal body cues. Hunger and satiety play an important role here, controlling food intake and maintaining nutrient homeostasis. In C. elegans, chemosensory neurons sense food and relay information to the rest of the animal via hormones, neurotransmitters, and neuropeptides. These responses have a direct impact in worms’ behaviour and physiology. This study shows that SKN-1B, an ortholog of the mammalian NF-E2 related transcription factors (Nrfs), acts in the two hypothalamus-like ASI chemosensory neurons to sense food, communicate nutritional status to the organism, and control food-related behaviours. SKN-1B modulates IIS and TGF-β pathways to suppress satiety-induced quiescence and promote exploratory behaviour. Finally, SKN-1B influences physiology by promoting a robust mitochondrial network which is required for energy homeostasis. The work presented here, suggests an exciting role for mammalian Nrf proteins in food-sensing and satiety

Neuroprotektive Wirkung von Sideritis scardica Griseb. in Caenorhabditis elegans als Modellorganismus für neurodegenerative Erkrankungen

Sideritis scardica Griseb., also known as Greek mountain tea, is a plant that belongs to the family of Lamiaceae. It is endemic to the Balkan region, where the herb is traditionally used against a broad range of afflictions, mostly as an infusion. Over the past few years, S. scardica has been scientifically investigated for the pharmacological properties it is said to have, especially focusing on the effects in the central nervous system, such as an improved cognitive performance and the reduction of β-amyloid plaques which are typical in Alzheimer's disease. Following this issue, the main goal of the present study was to examine whether extracts of Greek mountain tea counteract further pathomechanisms of this and other neurodegenerative disorders which are a rising health concern in our ageing society. For this purpose six hydroalcoholic S. scardica extracts of different lipophilicity, that were produced and phytochemically analysed by Finzelberg GmbH & Co. KG, were investigated for their neuroprotective activity in the model organism Caenorhabditis elegans. Additionally, six fractions of one of the most potent extracts, seven isolated pure compounds and extracts of some well-known medicinal plants were tested. The mid-polar extracts of S. scardica in particular, as well as the more lipophilic fractions and the phenylethanoid glycosides, were counteracting the aggregation or toxicity of β-amyloid peptides, α-synuclein, 6-hydroxydopamine, tau- and polyglutamine proteins in vivo, whereas they did not reveal any antioxidant properties in the nematodes. The spectrum of activities of S. scardica was especially comparable with that of Bacopa monnieri (Brahmi), a plant used in ayurvedic medicine. The alleviation of the neurotoxicity of the proteins and compounds make S. scardica extracts interesting for the preventive or therapeutic treatment of Alzheimer's, Parkinson's, Huntington's and other neurodegenerative diseases. The phenylethanoids could be identified as active constituents, although synergistic effects between extractives of different polarity remain probable and have to be taken into consideration. Also, a hypothetical mode of action could be postulated: the bonding of polyphenols to the peptides, directly inhibiting their aggregation to toxic oligomers or plaques and fibrils. In the present study, the activity of hydroalcoholic Sideritis scardica extracts against hallmarks of Alzheimer's disease could be affirmed and for the first time, positive effects on further neurotoxic substances could be shown, expanding the neuropharmacological profile of the Greek mountain tea. As some studies on vertebrates and humans assessing the influence on cognitive performance and proving the harmlessness have already been conducted, high-quality clinical studies investigating the preventive or therapeutic efficacy against Alzheimer's etc. should be performed

Investigating the roles of SKN-1B and RNA polymerase III in ageing

The diseases of ageing represent an increasingly severe threat to human health, and most societies are experiencing an unprecedented demographic shift towards an older population. In this thesis the nematode worm C. elegans is used to examine two aspects of the ageing process. Firstly, it investigates the function of skn-1b. skn-1 is the C. elegans orthologue of mammalian NRF proteins and is required for normal longevity and stress resistance. The skn-1b isoform is expressed in two ASI neurons and is known to be required for the longevity resulting from the life-extending intervention dietary restriction, however little is known about the mechanisms by which this is possible. This study shows that skn-1b expression peaks at the L2 stage and that its levels and patterns are modulated by food levels in the external environment with daf-11 being an important mediator of this. Signalling from ASI neurons via secreted ILPs and TGF-β ligand appears disrupted in animals lacking skn-1b, and animals lacking skn-1b display a cell non-autonomous failure to properly activate the UPRER when challenged with tunicamycin. It also reports that the SKN-1B protein is a functional transcription factor, but that skn-1b is not required for oxidative stress resistance, normal lifespan or brood size. In the second part of this thesis we identify RNA polymerase III as limiting lifespan in C. elegans, showing that knockdown of Pol III function using rpc-1 RNAi can extend lifespan either when active in the whole worm or just the intestine. Inhibition of Pol III protects the C. elegans intestine showing that at least one ageing pathology is delayed. Finally, this thesis shows that this lifespan extension is separable from that resulting from inhibition of 12 Ribosomal Protein S6 Kinase (rsks-1). Taken together, the findings presented here shed more light on the role of transcriptional regulators in the ageing process