Li, Weixun et al. (2011) International Worm Meeting "Differential roles of thioredoxin reductases in C. elegans."

Bandyopadhyay, Jaya, Lee, Sunkyung, Cho, Jeong Hoon, Li, Weixun, Ahnn, Joohong

[International Worm Meeting, 2011]

Thioredoxin reductases are members of flavoprotein family of pyridine nucleotide-disulfide oxidoreductases. These enzymes use NADPH to reduce thioredoxin, one of the major protein disulfide reductases serving as electron donors for various biological substrates.In this study, we report that two thioredoxin reductases in C. elegans, cytosolic trxr-1 and mitochondrial trxr-2. GFP reporter system revealed that trxr-1 ismainly expressed in intestine whereas trxr-2 is expressed in muscle system. trxr-2 mutants weremore sensitive toparaquatthan trxr-1mutants in terms of lethality and developmental delay, and was rescued by overexpression of wild type TRXR-2. Interestingly, the activity of V-ATPase, a proton pump residing at acidic intracellular organelles, was reduced in trxr-1deletion and trxr-2;trxr-1 double mutants, but not in trxr-2 mutants. We present evidence that V-ATPase activity is independent of thioredoxin system. These results suggest that spatial separation of trxr-1 and trxr-2 confer them with differential roles in oxidative stress defense and regulation of V-ATPase activity.

Li, Weixun et al. (2009) International Worm Meeting "Functional Studies on Thioredoxin Reductases in C. elegans."

Li, Weixun, Cho, Jeong Hoon, Ji, Yon Ju, Cho, Cha-Sun, Bandyopadhyay, Jaya, Lim, Yun-Ki

[International Worm Meeting, 2009]

Thioredoxins have been described as small (~12kDa), and ubiquitous disulfide reductases. Thioredoxins are multifunctional proteins; the anti-oxidative activity is one of the most well studied functions. Previously one of the thioredoxin (trx-1) in C. elegans has been characterized in our laboratory. The reducing activity of thioredoxin has been demonstrated in vitro. The trx-1 has been found to express in head neurons and intestine. Null mutant of trx-1, has been found to show shorter life span than that of wild type, and showed less viability under stress condition. Further, these defects of mutant can be completely rescued by transforming full length construct of trx-1into mutants. These results showed that thioredoxin as an anti-oxidative protein, is important for maintaining the normal life span and surviving under stress condition. Thioredoxin reductases are enzymes belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases. These enzymes use NADPH to reduce thioredoxin. There are two thioredoxin reductases in C. elegans, trxr-1 and trxr-2. The trxr-1 is predicted to localize in cytoplasm, whereas trxr-2 is predicted to localized mitochondria.GFP reporter system revealed that both trxr-1 and trxr-2 are expressed in intestine the same as trx-1. Consistent with prediction and biochemical analysis, we found that trxr-2 showed mitochondrial GFP expression pattern. Further, ectopic expression of trxr-2 in muscle confirmed the mitochondrial localization of trxr-2. Interestingly, trxr-2 seems to be induced by stress and knockout mutants of trxr-2 were less viable under stress condition. These results suggest that trxr-2 is mainly involved in stress response whereas trxr-1 may be response for other biological phenomena. Further studies of these two genes are under investigation.

Li, Weixun et al. (2009) International Worm Meeting "The Effects of Manganese and Mn Homeostasis on Pathogen Infection in C. elegans."

Li, Weixun, Cho, Jeong Hoon

[International Worm Meeting, 2009]

Nramp1 (Natural resistance-associated macrophage protein-1) is a manganese transporter in macrophage functionally conserved in bacteria and eukaryotes. Manganese (Mn), which is required in small amounts functions as a cofactor for most antioxidants, and also known as a superoxide scavenger. It is also known that mouse defective in Nramp1 is susceptible to pathogen. Three Nramp homologues, smf-1, smf-2, and smf-3, have so far been identified in the nematode, Caenorhabditis elegans. GFP promoter assay revealed intestinal expressions of smf genes from early embryonic stages and these expressions were maintained through the adult stages. Furthermore, smf deletion mutants and smf RNAi have increased sensitivity to oxidative stress, EDTA and excess manganese. Interestingly, defective SMFs cause hypersensitivity to the pathogen, Staphylococcus aureus but not to the non-pathogenic Bacillus subtilis, indicating that manganese uptake is essential for the innate immune system. We are further investigating the roles of Manganese and other Mn homeostasis related genes, such as the Golgi calcium/manganese ATPase, pmr-1, and the vacuolar H+-ATPase, vha-8 in the worms'' defense system. We propose that C. elegans intestinal lumen provide a good and a simple model for macrophage phagosome especially in Mn-mediated innate immune systems.

Li, Weixun et al. (2015) International Worm Meeting "Regulator of Calcineurin (RCAN-1) in AFD neurons regulates thermotaxis behavior."

Lee, Sun-Kyung, Bell, Harold, Ahnn, Joohong, Li, Weixun

[International Worm Meeting, 2015]

Regulator of calcineurin (RCAN) is a calcineurin-interacting protein that inhibits calcineurin phosphatase. In this study, we found that rcan-1 deletion mutants exhibited cryophilic behavior dependent on tax-6, which was rescued by expressing rcan-1 in AFD neurons. Interaction between RCAN-1 and TAX-6 requires the conserved PxIxIT motif of RCAN-1, without which thermotactic behavior could not be fully rescued. Calcium imaging studies indicate that AFD neurons in rcan-1 and tax-6 mutants are abnormal. In addition, the loss of crh-1/CREB suppressed the thermotaxis phenotypes of rcan-1 and tax-6 mutants, suggesting that crh-1 may play a role downstream of the rcan-1/tax-6 pathway. Taken together, our results suggest that rcan-1 is an inhibitory regulator of tax-6 in AFD neurons, and that it acts in the formation of thermosensory behavioral memory in C. elegans. .

Weixun Li et al. (2010) East Asia Worm Meeting "Two thioredoxin reductase isoforms in C.elegans"

Jaya Bandyopadhyay, Weixun Li, Joohong Ahnn, Jeong Hoon Cho, Sunkyung Lee

[East Asia Worm Meeting, 2010]

Thioredoxin reductases are members of flavoprotein family of pyridine nucleotide-disulfide oxidoreductases. These enzymes use NADPH to reduce thioredoxin, a major protein disulfide reductase serving as electron donors for various reducing enzymes including peroxiredoxin scavenging H2O2. Therefore, this thioredoxin system is critical for control of redox status of cellular system. In this study, we report that two thioredoxin reductases in C. elegans, trxr-1 and trxr-2. GFP reporter system revealed that both trxr-1 and trxr-2 are expressed in intestine as trx-1, a cytoplasmic thioredoxin of C. elegans. Mitotracker staining revealed that trxr-1 was localised in cytoplasm, and trxr-2 in mitochondria as predicted by their signal sequence analysis. Both trxr-1 and trxr-2 mutants showed retarded growth when exposed to paraquat. Interestingly, V-ATPase activity was reduced in trxr-1 and trxr-1;trxr-2 double mutants, while it was comparable to wildtype in trxr-2 mutant. These results suggest that spatial separation of trxr-1 and trxr-2 confer them with differential roles in oxidative stress defense and regulation of enzymatic activity. Oxidized protein levels, transcriptional regulation, and lifespans in different conditions of these genes are currently under investigation.

Gopal E et al. (2015) J Pharmacol Exp Ther "Species-specific influence of lithium on the activity of SLC13A5 (NaCT): lithium-induced activation is specific for the transporter in primates."

Ganapathy V, Ramachandran S, Gopal E, Babu E, Prasad PD, Bhutia YD

[J Pharmacol Exp Ther, 2015]

NaCT (SLC13A5) is a Na(+)-coupled transporter for Krebs cycle intermediates and is expressed predominantly in the liver. Human NaCT is relatively specific for citrate compared with other Krebs cycle intermediates. The transport activity of human NaCT is stimulated by Li(+), whereas that of rat NaCT is inhibited by Li(+). We studied the influence of Li(+) on NaCTs cloned from eight different species. Li(+) stimulated the activity of only NaCTs from primates (human, chimpanzee, and monkey); by contrast, NaCTs from nonprimate species (mouse, rat, dog, and zebrafish) were inhibited by Li(+). Caenorhabditis elegans NaCT was not affected by Li(+). With human NaCT, the Li(+)-induced increase in transport activity was associated with the conversion of the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type. H(+) was able to substitute for Li(+) in eliciting the stimulatory effect. The amino acid Phe500 in human NaCT was critical for Li(+)/H(+)-induced stimulation. Mutation of this amino acid to tryptophan (F500W) markedly increased the basal transport activity of human NaCT in the absence of Li(+), but the ability of Li(+) to stimulate the transporter was almost completely lost with this mutant. Substitution of Phe500 with tryptophan in human NaCT converted the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type, an effect similar to that of Li(+) on the wild-type NaCT. These studies show that Li(+)-induced activation of NaCT is specific for the transporter in primates and that the region surrounding Phe500 in primate NaCTs is important for the Li(+) effect.

Tabuse Y (1997) International C. elegans Meeting "LITHIUM CAUSES DEVELOPMENTAL DISORDER IN C. ELEGANS"

Tabuse Y

[International C. elegans Meeting, 1997]

Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to be a most effective treatment of manic-depressive illness.!@Although several models have been proposed to explain Li action, the molecular mechanism has remained unclear. Recently, GSK (glycogen synthase kinase)-3b was proposed to be a target of Li, suggesting that Li affects the wnt signaling pathway. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 oC. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants that propagated on Li-containing medium. Several mutants were isolated, and one of them was mapped on the left side of unc-42 on chromosome V. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. So, this mutation appeared to be maternal. Further genetic analyses of the mutants and the observation on the cellular phenotype of Li-treated embryos are underway.

Tabuse Y (1996) Worm Breeder's Gazette "Lithium arrests embryonic development of C. elegans"

Tabuse Y

[Worm Breeder's Gazette, 1996]

Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to bea most effective treatment of manic-depressive illness. Although several models have been proposed to explain Li action, the molecular mechansm remains unclear. The most widely accepted model is the inositol depletion hypothesis, in which Li is thought to affect inositol phosphate turnover by inhibiting inositol monophosphatase, thus resulting in the depletion of endogenous inositol. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 C and observed the laid embryos. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants, which propagated on Li-containing medium. So far, I obtained one mutant. The mutation was tentatively assigned to chromosome V. Preliminary genetic analysis showed that the mutation showed maternal effect. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. I am now trying to isolate other mutants and also to identify early defects of embryogenesis caused by lithium treatment. I would like to thank J. Miwa for encouragement and discussions.

Inokuchi A et al. (2015) J Appl Toxicol "Effects of lithium on growth, maturation, reproduction and gene expression in the nematode Caenorhabditis elegans."

Matsusaki H, Yamamoto R, Takumi S, Morita F, Inokuchi A, Arizono K, Tominaga N, Ishibashi H

[J Appl Toxicol, 2015]

Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000m, respectively, for LiCl and 750, 750 and 3000m, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78m LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375m Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans.

McColl G et al. (2008) J Biol Chem "Pharmacogenetic analysis of lithium-induced delayed aging in Caenorhabditis elegans."

Melov S, McColl G, Lithgow GJ, Vantipalli MC, Killilea DW, Hubbard AE

[J Biol Chem, 2008]

Lithium (Li+) has been used to treat mood affect disorders, including bipolar, for decades (1;2). This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the underlying mechanism(s) of its therapeutic action is not understood. Here we describe a pharmacogenetic study of Li+ in the nematode Caenorhabditis elegans. Exposure to Li+ at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li+ treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference (RNAi) of T08D10.2 is sufficient to extend longevity (~25% median increase), suggesting Li+ regulates survival by modulating histone methylation and chromatin structure.