Yasuaki Saitoh et al. (2010) East Asia Worm Meeting "Study on physiological functions of D-amino acid degradative enzymes in nematode Caenorhabditis elegans"

Yousuke Seida, Kazuhiro Maeda, Tomonori Kawata, Masumi Katane, Hiroyuki Kobuna, Takao Inoue, Yasuaki Saitoh, Hiroyuki Arai, Yasuhito Nakagawa, Masae Sekine, Taro Sakamoto, Hiroshi Homma, Takemitsu Furuchi

[East Asia Worm Meeting, 2010]

Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser has been proposed as a neuromodulator that regulates L-glutamate-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating developmental processes, hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, while DDO is highly specific for acidic D-amino acids. DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively, as well as mediate the elimination of accumulated exogenous D-amino acids in various organs. Previously, we demonstrated that nematode Caenorhabditis elegans, a multicellular model animal has at least one active DAO gene and three active DDO genes, while it had been thought that most organisms bear only one copy of each DAO and DDO gene. In addition, our previous study revealed that the spatiotemporal distributions of these enzymes in the body of C. elegans are different from one another. In this study, to elucidate the physiological roles of the C. elegans DAO and DDOs, we characterized several phenotypes of four C. elegans mutants in which each gene is partially deleted and inactivated. We also determined free D-amino acid contents in several worm samples using high-performance liquid chromatography (HPLC) techniques. We will report the phenotypes of the C. elegans mutants in comparison with those of wild-type C. elegans, as well as alterations in D-amino acid levels within the body.

Saitoh, Yasuaki et al. (2013) International Worm Meeting "D-Aspartate oxidase is involved in the caloric restriction-induced lifespan extension in C. elegans."

Inoue, Takao, Sekine, Masae, Saitoh, Yasuaki, Arai, Hiroyuki, Furuchi, Takemitsu, Sakamoto, Taro, Okutsu, Mari, Homma, Hiroshi, Katane, Masumi

[International Worm Meeting, 2013]

Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most intensively studied. In mammals, D-Ser has been proposed as a neuromodulator that regulates L-glutamate (L-Glu)-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. Mammalian DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively. Previously, we demonstrated that D-Ser, D-Asp, D-Glu and D-alanine (D-Ala) are present in nematode Caenorhabditis elegans, a multicellular model animal. We also found that C. elegans has at least one active DAO gene and three active DDO genes (DDO-1, DDO-2 and DDO-3), and that the spatiotemporal distributions of these enzymes in the body of C. elegans differ from one another. Furthermore, our previous study showed that alterations of brood size and hatching rate are observed in four C. elegans mutants lacking each gene for the DAO and DDOs. Interestingly, lifespan extension was observed in the DDO-3 mutant. To characterize the mechanism of lifespan extension in the DDO-3 mutant, we performed genetic epistasis experiments to test interactions between the DDO-3 gene and other known longevity pathways. The results suggest that DDO-3 is involved in caloric restriction-induced lifespan extension but not in insulin/IGF signaling pathway, NAD/sir2 pathway nor mitochondrial electron transport system. We also found that D-Glu and L-tryptophan (L-Trp) accumulate throughout life in the DDO-3 mutant. Now we are investigating the relationship between aging and the accumulations of D-Glu and L-Trp.

Qiu, Tian (Autumn) et al. (2021) International Worm Meeting "Bacterial D-alanine metabolism affects C. elegans lifespan under high glucose conditions"

Qiu, Tian (Autumn), Schroeder, Nathan, Sweedler, Jonathan

[International Worm Meeting, 2021]

Microbiome-derived metabolites are able to impact the host's nervous system through the gut-brain axis. D-amino acids (D-AAs), the D-enantiomers of prevalent L-amino acids, have unique functions such as neuromodulators in animals including mollusks, rodents, and primates, while microbiota is a known contributor to the source of D-AAs in animals. Among all D-AAs, D-Ala is a potent agonist of the glycine binding site of N-methyl-D-aspartate (NMDA) glutamate receptors in vitro. Additionally, D-Ala immunoreactivity in pancreatic beta-cells and adrenocorticotropic hormone (ACTH)-secreting cells suggests its involvement in glucose homeostasis. While several researches showed microbiota as a major source of D-Ala in animals, the effect of bacterial D-Ala synthesis and metabolism on host physiology has not been studied. In this study, we fed wild type Caenorhabditis elegans N2 with Escherichia coli mutants with knockout of genes relevant to D-Ala biosynthesis (dadX, alr) and D-Ala metabolism (ddlA), and then examined changes in C. elegans phenotypes with or without the presence of high glucose. All three bacterial mutants displayed similar growth curves in nutrient-rich liquid media, but decreased D-Ala/total alanine ratio compared to the parental E. coli strain. To prevent the introduction of exogenous D-Ala, we cultured C. elegans on peptone-free NGM media. We found no statistically significant differences in life span when fed on the selected E. coli mutants. However, we observed a slight avoidance to all three bacterial mutants compared to the parental strain. When amended with 40 mM glucose, deltaddlA significantly decreased the life span of N2. No food preference was observed on glucose-amended plates. These results indicate that the combination of deficiency in bacterial DdlA activity and high glucose led to a decreased life span in C. elegans.

Nektarios Tavernarakis et al. (2001) International C. elegans Meeting "Execution of necrotic-like cell death in C. elegans requires the activity of specific aspartyl proteases"

Monica Driscoll, Keli Xu, Nektarios Tavernarakis

[International C. elegans Meeting, 2001]

Distinct types of cell death underlie the pathology of many human diseases, yet very little is understood of non-apoptotic cell death mechanisms. Analysis of non-apopotic death mechanisms in C. elegans is likely to provide mechanistic insights relevant to critical problems in neuronal injury and pathology. Gain-of-function mutations in specific C. elegans ion channel genes encode hyperactive channels that induce necrotic-like death of the neurons that express these mutant genes (Chalfie and Wolinsky, 1990; Driscoll and Chalfie, 1991). For example, dominant mutations affecting the mec-4 degenerin channel induce degeneration of six touch receptor neurons. mec-4(d )-induced cell death occurs independently of all known regulators and executors of programmed cell death in C. elegans , and is necrotic-like in that it is characterized by dramatic cellular swelling. We are investigating the requirements for the progression and execution of degenerative cell death inflicted by mec-4(d) and other insults in C. elegans . Essentially nothing is known of the molecules that execute necrotic cell death, although it is clear that, like in apoptosis, proteolysis is likely to be a major component of the death mechanism. We have found that specific aspartyl proteases of the Cathepsin D family are required for the efficient execution of necrotic-like cell death in C. elegans . Several mutant nematode strains have been found to exhibit up to 90% reduced Cathepsin D protease activity (Jacobson et al., 1988). We assessed the requirement for Cathepsin D activity in necrotic-like cell death induced by mec-4(d) by testing cad-1 alleles j1 and j14 for the ability to block cell death. Necrotic-like cell death induced by mec-4(d) was significantly reduced in these genetic backgrounds. In addition, several mutations that have been reported to cause 80-90% reduced cathepsinD activity ( daf-4(e1364) , mec-8(e398) and unc-52(su250) also suppress mec-4(d)- induced degeneration. Starvation is another condition that leads to significant reduction of Cathepsin D activity in C. elegans (Sarkis et al. 1988). We have observed that starvation also interferes with necrotic-like cell death induced by mec-4(d) . To further demonstrate the involvement of Cathepsin D proteolytic activity in neurodegeneration, we assayed the effects of Pepstatin A, a potent and specific inhibitor of Cathepsin D on mec-4(d) induced necrotic-like cell death. Pepstatin A significantly blocked neurodegeneration. Thus, one or more cathepsin D proteases play a significant role in mec-4(d)- induced degeneration. There are several genes in the C. elegans genome that encode putative Cathepsin D proteases. The most highly similar to biochemically characterized mammalian aspartyl proteases has been designated asp-4 (Tcherepanova et al., 2000). We employed dsRNA interference to quench asp-4 expression and to determine the effects on neurodegeneration. Results indicate that ASP-4 activity is required for necrotic-like cell death induced by mec-4(d ). Conversely, overexpression of asp-4 in a mec-4(d);cad-1(j1) mutant completely reversed the suppressing effects of cad-1(j1) on mec-4(d) induced neurodegeneration. We tested all other Cathepsin D proteases in the C. elegans genome for similar effects on death when inactivated via RNAi or when over-expressed. Our analysis revealed that ASP-3 is the only other aspartyl protease required for necrotic-like cell death. This is the first clear genetic demonstration of a requirement for a protease in the execution of necrotic-like cell death in C. elegans . Aspartyl proteases such as Cathepsin D are mostly lysosomal enzymes but they have also been found to function in the cytoplasm. Interestingly, Cathepsin D has been implicated in endocytic degradation of proteins and mec-4(d)- induced cell death involves extensive plasma membrane internalization that resembles endocytosis. Such a process may require the function of Cathepsin D. Our results suggest that Cathepsin D activity plays a pivotal role in the destruction of the cellular contents during mec-4(d)- induced neurodegeneration. Preliminary results suggest that cathepsin D may also play a role in necrotic-like cell death induced by insults other than mec-4(d) in the nematode. Establishment of a central role for cathepsin D in the execution of degenerative cell death would make cathepsin proteases attractive targets for therapeutic intervention in human injury and disease.

Kwon, Namseop et al. (2013) International Worm Meeting "3-D Worm Tracker for C. elegans."

Lee, Seung-Jae, Je, Jung Ho, Kwon, Namseop, Hwang, Ara B.

[International Worm Meeting, 2013]

Various tracking systems have been developed to quantify the behavior of C. elegans, a genetic model organism for studying animal behaviors. The tracking systems, however, provide two dimensional (2-D) imaging and thus have limitations in understanding the behaviors of worms freely moving in 3-D environments. Here, we introduce a 3-D worm tracker (3DWT) that is capable of analyzing trajectories and postures of worms crawling in 3-D environments. The prototype 3DWT was based on a stereoscopic imaging system that consisted of two cameras aligned at right angles, recording stereoscopic images of a single worm moving in a 3-D sample chamber. From these two 2-D images, 3-D information such as the trajectories or the postures of the worm in 3-D environments was reconstructed by stereomatching. We found that the trajectories and the postures in 3-D environments are not restricted in a single 2-D plane. This result confirms the idea that the movements of C. elegans in 3-D environments are more complex than those of worms moving on agar plates. For analyzing the kinematics of C. elegans, we introduced "bending vector" that is defined by the magnitude and direction of the bending at each body part of a worm. The bending vectors with various directions propagated from head to tail during the worms' forward runs, suggesting that the bending vector can be a useful analytic parameter for understanding complex undulatory patterns of worms in 3-D environments. Currently, we are improving our 3DWT to automatically track a worm by recording the stereoscopic images on a single camera, instead of two cameras, using four mirrors. We are furthermore enhancing the throughput and accuracy of the reconstruction process by incorporating additional camera perpendicularly to the stereoscopic configuration. We believe that our 3DWT would provide new opportunities to analyze the behaviors of C. elegans in 3-D environments and would help understand its behaviors in nature.

Laura Bianchi et al. (2004) West Coast Worm Meeting "Calcium permeability of death-inducing DEG/ENaC ion channel MEC-4(d)"

Dewey Royal, Monica Driscoll, Beate Gerstbrein, Gargi Mukherjee, Jian Xue, Laura Bianchi

[West Coast Worm Meeting, 2004]

The C. elegans ion channel complex formed by the DEG/ENaC MEC-4 and MEC-10 subunits, exclusively assembled in six specialized touch sensing neurons, is thought to constitute the core of a voltage-independent Na + -selective mechanosensory ion channel. In a screen for touch insensitive nematodes, a very interesting mec-4 mutant was isolated. This mec-4(d) mutation specified a large side-chain amino acid substitution near the channel pore and induces degeneration of touch neurons through a mechanism that appears to involve channel hyperactivation . Analysis of mec-4(d)- induced neurodegeneration , however, indicated that large increase of [Ca 2+ ] i is one of the molecular requirements for cell death. While Ca 2+ release from ER-based stores is certainly occurring in mec-4(d) -induced cell-death, the triggering factor remains unknown. We speculated that Ca 2+ could permeate through MEC-4(d) channels to perhaps induce Ca 2+ -induced Ca 2+ release from stores. P> P> We analyzed Ca 2+ permeability of MEC-4(d) channels reconstituted in Xenopus oocytes and found that they are permeable to Ca 2+ ( P Ca /P Na ~ 0.22). Interestingly, Ca 2+ currents through MEC-4(d) channels display the same sensitivity to amiloride that Na + currents do, with Ki for the drug being reduced by co-expression with MEC-10(d). We also found that Ca 2+ permeability is independent of co-expression with MEC-10(d), MEC-2 or MEC-6, and appears to be determined by MEC-4(d) residues. We are currently searching for residues involved in Ca 2+ permeability, by introducing second site mutations in and near the pore, identified in our screen for suppressors of mec-4(d) -induced cell death. In addition we are evaluating the intracellular pathway activated by entry of Ca 2+ through MEC-4(d) that leads to massive release of Ca 2+ from the stores that ultimately results in cell death.

Sato, M. et al. (2013) International Worm Meeting "The rare sugar D-psicose extends Caenorhabditis elegans lifespan by increasing oxidative stress resistance."

Shintani, T., Sato, M., Okuma, K., Izumori, K., Sakoguchi, H.

[International Worm Meeting, 2013]

Calorie-restricted diets are known to extend lifespans of model animals, including nematodes, flies, mice and monkeys. It is believed that calorie-restricted diets delay the onset of aging-associated diseases such as diabetes, cancer and Alzheimer's disease, and also prolong human longevity. However, in reality, it is difficult to continue calorie-restricted diets for a long period of time. Therefore, the development of calorie restriction mimetics (CRMs) that have similar effects as calorie-restricted diets is of great interest. Our preliminary study showed that the rare sugar D-psicose, a stereoisomer of D-fructose, extended the lifespan of Caenorhabditis elegans. The findings suggest that D-psicose serves as a CRM in C. elegans. The extension of C. elegans lifespan under calorie-restricted diet conditions is thought to be dependent on the induction of oxidative stress-related proteins, including Cu/Zn-superoxide dismutase (SOD), Mn-SOD and catalase regulated by the insulin/IGF-I signaling pathway. We aimed to elucidate the molecular mechanism of the lifespan extension by D-psicose. Wild-type C. elegans N2 was used in this study. Treatment of worms with 28 mM D-psicose increased mean lifespan of N2 by ca. 20% (control: 20.9 days; D-psicose: 25.1days). Using Quantitative real-time RT-PCR, we found that the mRNA expression levels of mitochondrial Mn-SOD (sod-3), cytosolic catalase (ctl-1) and peroxisomal catalase (ctl-2) were enhanced by 1.4, 1.5 and 1.6-fold, respectively, after treatment of D-psicose. In contrast, expression of cytosolic Cu/Zn-SOD (sod-5) was unaffected by 28mM D-psicose treatment. Total SOD and catalase enzyme activities in N2 with 28 mM D-psicose treatment were increased by 1.4 and 2.1-fold, respectively. D-psicose was thought to extend the lifespan of C. elegans by increasing the oxidative-stress resistance of the nematode. These results indicate that D-psicose is a candidate CRM.

Laura Bianchi et al. (2004) East Coast Worm Meeting "Calcium permeability of death-inducing DEG/ENaC ion channel MEC-4(d)"

Jian Xue, Maryanne Royal, Laura Bianchi, Beate Gerstbrein, Wei-Hsiang Lee, Monica Driscoll, Gargi Mukherjee, Dewey Royal

[East Coast Worm Meeting, 2004]

The C. elegans ion channel complex formed by the DEG/ENaC MEC-4 and MEC-10 subunits, exclusively assembled in six specialized touch sensing neurons, is thought to constitute the core of a voltage-independent Na + -selective mechanosensory ion channel. In a screen for touch insensitive nematodes, a very interesting mec-4 mutant was isolated. This mec-4(d) mutation specified a large side-chain amino acid substitution near the channel pore and induces degeneration of touch neurons through a mechanism that appears to involve channel hyperactivation . Analysis of mec-4(d)- induced neurodegeneration , however, indicated that large increase of [Ca 2+ ] i is one of the molecular requirements for cell death. While Ca 2+ release from ER-based stores is certainly occurring in mec-4(d) -induced cell-death, the triggering factor remains unknown. We speculated that Ca 2+ could permeate through MEC-4(d) channels to perhaps induce Ca 2+ -induced Ca 2+ release from stores. P> P> We analyzed Ca 2+ permeability of MEC-4(d) channels reconstituted in Xenopus oocytes and found that they are permeable to Ca 2+ ( P Ca /P Na ~ 0.22). Interestingly, Ca 2+ currents through MEC-4(d) channels display the same sensitivity to amiloride that Na + currents do, with Ki for the drug being reduced by co-expression with MEC-10(d). We also found that Ca 2+ permeability is independent of co-expression with MEC-10(d), MEC-2 or MEC-6, and appears to be determined by MEC-4(d) residues. We are currently searching for residues involved in Ca 2+ permeability, by introducing second site mutations in and near the pore, identified in our screen for suppressors of mec-4(d) -induced cell death. In addition we are evaluating the intracellular pathway activated by entry of Ca 2+ through MEC-4(d) that leads to massive release of Ca 2+ from the stores that ultimately results in cell death.

Dhillon HS et al. (1995) International C. elegans Meeting "Toward Developing a mec-4(d) Ablation Vector"

Kinnell M, Dhillon HS, Driscoll MA, Lai CC

[International C. elegans Meeting, 1995]

mec-4(d) alleles (which encode substitutions of large sidechain amino acids for a conserved Ala residue at position 713 in the full length channel protein) induce swelling and degeneration of the touch receptor neurons, the cells in which mec-4 is normally expressed. We have expressed mec-4(d) ectopically and have found that many cell types are susceptible to the deleterious effects of mec-4(d). Expression of a genomic mec-4(d) gene from the mec-7 promoter induces degeneration of the touch receptor neurons and the other cells in which mec-7 is expressed, expression of mec-4(d) from the unc-54 promoter causes muscle hypercontraction and paralysis, and expression of mec-4(d) from hsp/16 results in widespread degenerative cell death. The toxic property of MEC-4(d) might be useful to C. elegans researchers. For example, induction of degeneration of specific groups of neurons could help verify identities of cells found to express a specific gene by causing predicted behavioral abnormalities (A. Maricq and C. Bargmann successfully used this approach). With the considerable progress of the Genome Sequencing Project, many genes will be identified that will not be correlated with mutant phenotypes. Introducing 5' regions from such genes to drive expression of mec-4(d) could help in deducing both the expression pattern of the gene and a potential mutant phenotype (provided that activity of the gene in question is essential to cell function). We have constructed mec-4(d) genomic and cDNAs that can be used for this purpose and are in the process of constructing an ablation vector that will facilitate promoter fusions.

Lori Langiano et al. (2002) Mid-west Worm Meeting "Analysis of a Proposed Cell Binding Site on C. elegans Collagen IV."

Lori Langiano, Colette M Witkowski

[Mid-west Worm Meeting, 2002]

Type IV collagen, one of the most abundant extracellular matrix proteins, provides structural support, developmental cues, and contains cell binding sites (CBS) for cellular receptors. Most collagen IV molecules are composed of a heterotrimer formed by two alpha 1 chains (emb-9) and one alpha 2 chain (let-2), with CBS domains in the triple helix region. The human collagen IV molecule contains a CBS composed of aspartate (D) on each alpha 1 chain and arginine (R) on the alpha 2 chain. These form a three-dimensional D-R-D sequence that is recognized by the cell surface receptor, integrin alpha 1 beta 1. Amino acid sequence alignment has revealed that the D-R-D sequence is conserved on the collagen IV molecule of C. elegans. This proposed CBS sequence is being analyzed to determine its function. Previous data suggest this site has potential importance during C. elegans development.