Young-Ki Paik et al. (2005) International Worm Meeting "Role of Daumone and Cholesterol in the Metabolic Aging of Caenorhabditis elegans"

Young-Ki Paik, David J Chitwood, Mankil Jung, Heekyeong Kim, Yong-hyeon Yim, Pan-Young Jeong

[International Worm Meeting, 2005]

We will present data on two key metabolic regulators of C. elegans: dauer-inducing pheromone (daumone) and cholesterol. First, we will focus on daumone, which signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle with distinctive adaptive features and extended lifespan. To elucidate a daumone-mediated signaling pathway, we isolated natural daumone from C. elegans by large-scale purification, then synthesized and characterized daumone, which was found to be a novel glycolipid (ascarylose-fatty acid). We will show that both natural and chemically synthesized daumones equally induce dauer larva formation in C. elegans (N2 strain) and certain dauer mutants, and also result in competition between food and daumone. Second, we will discuss a role of cholesterol in metabolic aging. C. elegans, lacking the de novo sterol biosynthetic pathway, requires sterol as an essential nutrient. We microinjected a human 7-dehydrocholesterol reductase (DHCR) expression vector into C. elegans, which was then chromosomally incorporated by 47;-radiation. This transgenic C. elegans (cholegans, i.e., cholesterol producing C. elegans) showed 31% longer lifespan when grown in sterol-deficient medium and contained 80% more cholesterol than wild-type control. Brood size of cholegans was reduced 40% compared to wild-type control, although growth rate was not significantly altered. The biochemical basis for lifespan extension of cholegans appears to partly result from its acquired resistance against both UV irradiation and thermal stress. Taken together, our results should help to elucidate roles for daumone and cholesterol in ageing. (Supported by BioGreen21 Project [to YKP])

Young-Ki Paik et al. (2006) East Asia C. elegans Meeting "A Comprehensive Proteomic Analysis of Dauer and Cholesterol Signaling Pathway in Caenorhabditis elegans"

Keun Na, Mi Jeong Jeong, Pan-Young Jeong, Young-Ki Paik

[East Asia C. elegans Meeting, 2006]

With the availability of its complete genome sequence and unique biological features relevant to human disease, C. elegans has become an invaluable model organism for the studies of proteomics, leading to the elucidation of nematode gene function. We have focused on two key metabolic regulators of C. elegans: dauer-inducing pheromone (daumone; Jeong et al., 2005, Nature, 433, 541-545) and cholesterol (Choi et al, 2003, MCP, 2, 1086-95). First, using various proteomic tools including 2D-LC-MS/MS and DIGE, we analyzed proteomic profiles upon treatment of daumone, which signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle. We found that there are multi-level changes in protein expression in the area of energy metabolism (decreased) and oxidative defense system (increased), suggesting that differential changes in various metabolic pathways appear to accommodate this non-aged state. This proteomic data was also paralleled with DNA microarray studies, which provided a window for direct comparison of proteomic and genomic profiles in dauer state. Second, we have launched a global C. elegans proteome project (CEPP) in order to analyze a comprehensive map of proteins involved in cholesterol signaling pathway in embryonic stage of C. elegans. In this talk, we will discuss a few lessons as well as some progress obtained from the pilot phase with respect to data management and functional annotation in C. elegans

Y Kimura et al. (1999) International C. elegans Meeting "Ca2+/CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans"

H Tokumitsu, M Muramatsu, K Eto, Y Kimura

[International C. elegans Meeting, 1999]

In mammals, Ca 2+ /CaM protein kinases (CaM-K) is thought to be important for Ca 2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named Ce CaM-KK and Ce CaM-KI, respectively) and analyzed their biochemical activities. Ce CaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, Ce CaM-KK activated mammalian CaM-KIV in vitro . Ce CaM-KI is highly homologous to mammalian CaM-KI and is activated by Ce CaM-KK through phosphorylation of Thr179 in a Ca 2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, Ce CaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH 2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of Ce CaM-KK and Ce CaM-KI cascade in vivo . Analyses for the expression patterns and isolation of mutant worms for both genes are underway.

Hahm, Jeong-Hoon et al. (2009) International Worm Meeting "A Molecular Mechanism for cGMP-Mediated Differential Production of Neuronal Insulin, Leading to Changes in Adult Longevity of Caenorhabditis elegans."

Kim, Sunhee, Hahm, Jeong-Hoon, Paik, Young-Ki

[International Worm Meeting, 2009]

G-proteins, including GPA-3, play an important role in regulating physiological responses in Caenorhabditis elegans. When confronted with an environmental stimulus such as dauer pheromone, or poor nutrients, C. elegans receives and integrates external signals through its nervous system (i.e., amphid neurons), which interprets and translates them into biological action. Here we show that a suppressed neuronal cGMP level caused by GPA-3 activation leads to a significant increase (47.3%) in the mean lifespan (MLS) of adult C. elegans through forkhead transcription factor family O (FOXO)-mediated signal. A reduced neuronal cGMP level was found to be caused by an increased cGMP-specific phosphodiesterase activity at the transcriptional level. Our results using C. elegans mutants with specific deficits in TGF-b and FOXO RNAi system suggest a mechanism in that cGMP, TGF-b and FOXO signaling interact to differentially produce the insulin-like molecules, ins-7 and daf-28, causing suppression of the insulin/IGF-1 pathway and promoting lifespan extension. Our findings provide not only a new mechanism of cGMP-mediated induction of longevity in adult C. elegans but also a possible therapeutic strategy for neuronal disease, which has been likened to brain diabetes.

Park, Saeram et al. (2015) International Worm Meeting "Deficiency in peroxisomal fatty acid metabolism causes abnormalities in Insulin/IGF-1 signaling and ER homeostasis, leading to an altered life history in Caenorhabditis elegans."

Joo, Hyoe-Jin, Park, Saeram, Paik, Young-Ki

[International Worm Meeting, 2015]

Metabolic regulation of fats is important for maintaining energy balance and healthy life in animals. Disturbance in balance between energy intake and expenditure causes obesity which accompanies type II diabetes and metabolic syndromes. Peroxisomal fatty acid beta-oxidation, one of the fatty acid metabolic processes, is also required for normal development in mammals, as seen in the cases of Zellweger syndromes and X-linked adrenoleukodystrophy. In Caenorhabditis elegans, defective fatty acid beta-oxidation in peroxisome causes accumulation of very long chain fatty acids in the form of fat granules, which resembles the obese state in mammals. Using the nematode C. elegans, a genetically well-established model animal for fat regulation, we investigated the effect of endogenously increased fat accumulation due to the deficiency of this pathway on the regulation of developmental processes and stress responses. Given that regulation of peroxisomal fatty acid beta-oxidation is critical for pheromone (ascaroside) biosynthesis in C. elegans, we were keen to examine the relationship between these two metabolic processes. Interestingly, the mutant animals with deficiency in the former such as daf-22(ok693) are less sensitive to the exogenous pheromones and exhibit reduced dauer formation. To elucidate the mechanism underlying this phenomenon, we checked insulin/IGF-1 signaling (IIS) pathway that negatively controls dauer formation. As a result, daf-22(ok693) exhibited reduced lifespan and sod-3 expression that are dependent on IIS, indicating the compromised DAF-16/FOXO activity. The expression of daf-28, one of insulin-like peptides, was also increased in daf-22(ok693), which might be responsible for both the compromised DAF-16/FOXO activity and the attenuated dauer formation in the presence of pheromones. Moreover, the expression of hsp-4, an IRE-1/XBP-1 dependent endoplasmic reticulum (ER) stress marker, was elevated in daf-22(ok693). Taken together, these results suggest that the fatty acid metabolism in peroxisome and ER homeostasis appear to be interlinked and involved in the maintenance of cellular signaling and life history of C. elegans.

Kimura Y et al. (2000) Japanese Worm Meeting "CaM-K-mediated signal cascade is conserved in some neurons of C. elegans in vivo"

Tokumitsu H, Kimura Y, Eto K

[Japanese Worm Meeting, 2000]

Ca2+/CaM protein kinases (CaM-K) is kown to have important roles for Ca2+-dependent signal transduction. CaM-K-mediated signal cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In addition, a transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation with downstream CaM-KIV. We characterized C. elegans orthologues of CaM-KK (ckk-1) and CaM-KI (cmk-1) and found that CaM-KK-CaM-KI-CREB cascade is conserved and operated both in vitro and in transfected cells. CMK-1is highly homologous to mammalian CaM-KI and is activated by CKK-1 through phosphorylation of Thr179 in a Ca2+ /CaM-dependent manner. An activated CMK-1 can phosphorylate CREB protein and activate its transcription activity. We constructed ckk::gfp and cmk::gfp fusion genes and analyzed the expression patterns of both genes. A ckk-1::gfp reporter was expressed small number of head neruons after L3 stage. On the other hand, cmk-1::gfpwas seen in the most of the neurons from embryo to adult. To see the localization of activated CREB in vivo, we made a reporter worm that conains 4XCRE::gfp fusion gene. GFP expression cannot be seen in normal development but the expression in small number of neurons in head and tail can be induced by cAMP soaking. This induction can also occur by the expression of constitutively active form of CMK-1. Interestingly, this expression can be seen only after L3 stage and the expression was decreased or disappeared in adult. We also found that the constitutively active form of CMK-1 induced the malformaion of vulva and Egl phenotype. These results suggest that CaM-KI-CREB cascade of C. elegans is also conserved in vivo but it works only in small number of neurons.

Cheong, Mi Cheong et al. (2011) International Worm Meeting "NSBP-1, a novel sterol binding protein, mediates the insulin/IGF-1 signaling pathway in response to cholesterol in Caenorhabditis elegans."

Paik, Young-Ki, Cheong, Mi Cheong, Na, Keun, Lee, Hyoung-Joo, Joo, Hyoe-Jin

[International Worm Meeting, 2011]

Here we investigated how cholesterol influences the Insulin/IGF-1 signaling (IIS) pathway in Caenorhabditis elegans. Cholesterol affects many IIS functions, including dauer formation rate, oxidative stress resistance, fat storage, and longevity. Moreover, DAF-16 transcriptional activity in IIS is also modulated by cholesterol. These responses are mediated by a novel protein called nematode sterol binding protein (NSBP)-1, which is a homolog of human nucleosome assembly protein 1 encoded by D2096.8. NSBP-1 is expressed at sites of cholesterol accumulation and translocates to the nucleus independent of DAF-16 in response to IIS. Site-directed mutagenesis revealed possible NSBP-1 phosphorylation at Thr203. Co-immunoprecipitation studies demonstrated that NSBP-1 associates with DAF-16 and that this interaction can be affected by cholesterol concentration. Experiments using RNAi against NSBP-1 and DAF-16 demonstrated that these proteins work together to regulate the effects of cholesterol on the IIS pathway, as well as cholesterol homeostasis, in C. elegans. Our results may be applicable to screening of novel therapeutic targets for ameliorating insulin-resistant diabetes which can be caused by hyperactivation of FOXO in mammals.

Lee, Daehan et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "TO RIDE OR NOT TO RIDE: GENETIC BASIS FOR NATURAL VARIATION OF C. elegans HITCHHIKING BEHAVIOR"

Lee, Junho, Kim, Heekyeong, Kruglyak, Leonid, Andersen, Erik C., Paik, Young-Ki, Lee, Daehan

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

In parasitic nematodes, cruising (widely foraging) and ambushing (sit-and-wait) are two main dispersal strategies for survival and host finding. Free living nematode use similar strategies. Given harsh conditions, Caenorhabditis elegans develops to a dauer larva, which can either cruise or display an ambushing behavior called nictation. This behavior may facilitate dispersal of dauers to new habitats in the wild environment through phoresy - using other animals as a transportation vehicle. Therefore, nictation may greatly affect the survivorship and reproduction of C. elegans. However, this ambushing strategy decreases the probability of finding new habitats by the cruising strategy. Thus, the decision making between cruising and nictating might be selected in nature. In this work, we characterized natural variation in nictation behavior among C. elegans wild isolates. By linkage mapping, we identified a major-effect quantitative trait locus (QTL) and a candidate gene for nictation behavior. This QTL might underlie natural variation of the C. elegans hitchhiking behavior in the wild. Our work clearly defines a specific QTL that may have an important evolutionary role by modulating decision making probability for a specific behavior.

Yoshishige Kimura et al. (2001) International C. elegans Meeting "CaM-K signal cascade of C. elegans functionsin vivo"

Hiroshi Tokumitsu, Masatoshi Hagiwawa, Yoshishige Kimura

[International C. elegans Meeting, 2001]

Ca 2+ /CaM protein kinases (CaM-K) have significant roles for Ca 2+ -dependent signal transduction. CaM-K-mediated signal cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). A transcription factor cAMP-responsive element binding protein (CREB) is known to be activated by phosphorylation with downstream CaM-KIV. In this study, we identified C. elegans orthologues of mammalian CaM-KK (ckk-1 ), CaM-KI ( cmk-1 ) and CREB, and found that CaM-KK-CaM-KI-CREB cascade is conserved and operated both in vitro and in transfected cells. Furthermore, we investigated the physiological functions through this signal cascade by genetical approaches. The distribution of activated CREB was analyzed by using the transgenic worms that carries 4XCRE::gfp fusion gene. GFP fluorescence cannot be seen in normal development, however, transgenic worms carrying constitutive-active form of cmk-1 induces GFP-expression in small number of sensory neurons in head and tail. On the other hand, wild type and mutant cmk-1 at T179A did not show any CREB activation, suggesting the requirement of Ca 2+ mobilization and the upstream kinase which was identified as CaM-KK . In addition, CREB-deficeient worms show no GFP fluorescence reasonably. Interestingly, when the worms were maintained in starved condition, the number of GFP-positive neurons increased and the intensity of GFP fluorescence was dramatically enhanced. These results suggest that CaM-KK-CaM-KI-CREB cascade of C. elegans is conserved in vivo in the some sensory neurons and activated by the circumstantial stimuli.

Park, Jun Young et al. (2019) International Worm Meeting "Functional cross-antagonism between opioid and pheromone signaling in stress avoidance in Caenorhabditis elegans."

Cheong, Mi Cheong, Paik, Young-Ki, Cho, Jin-Young, Koo, Hyun-Sook, Park, Jun Young

[International Worm Meeting, 2019]

When animals encounter stress, depending on the situation their best option might be to avoid it. Upon sensing starvation stress, Caenorhabditis elegans larvae (L2) elicit two opposing behaviors to escape from the stressful conditions: food-seeking roaming, mediated by NLP-24 opioid signaling, and dauer formation, mediated by ascaroside pheromone (or daumone) signaling. Since both signals originate in the ASI neurons, we hypothesized that they might mutually influence each other during starvation stress. Here, we show that the two pathways do indeed mutually influence each other and that their combined effects culminate in stress avoidance. In early L2 larvae, pheromones and their associated receptor-mediated signaling pathways negatively modulate nlp-24 expression in the ASI neurons. Likewise, the NPR-17 opioid receptor, suppresses pheromone biosynthesis, and overexpression of two opioid genes disturbs pheromone-mediated dauer formation. Interestingly, cGMP signaling appears to act as a switchboard that prioritizes these signals to manage stress avoidance behaviors depending on the duration of the starvation. During short-term starvation stress (STS), cGMP signaling appears to stimulate an opioid-mediated food-seeking behavior, whereas during long-term starvation stress (LTS), it stimulates pheromone-mediated dauer formation. Insulin and serotonin signaling differentially regulate nlp-24 expression in the ASI neurons, whereas a stress-induced isoform of SKN-1 and the TGF-?-related transcription factor NHR-69 suppress opioid signaling during LTS. Taken together, our results reveal a previously unexplored cross-antagonism between these two anti-stress signaling pathways that facilitates stress avoidance. Furthermore, our results suggest that opioid-overexpressing transgenic animals may be useful for studying the profound socio-economic problem of opioid overdose. [This study was supported by a grant from the National Research Foundation of Korea (2017R1A2B3003200 and 2011-0028112 to Y.-K.P).]