Rakow TL et al. (1995) International C. elegans Meeting "NEURONAL EXPRESSION OF UNC-13"

Bender J, Rakow TL, Maruyama IN, Brenner S

[International C. elegans Meeting, 1995]

The unc-13 gene has been determined to affect the nervous system of C. elegans. In unc-13 mutants, there is slow, irregular pharyngeal pumping, uncoordinated movement, and an insensitivity to cholinesterase inhibitors with an accumulation of acetylcholine. The Unc-13 protein contains a diacylglycerol binding domain, consisting of cysteine repeats and a calcium, phospholipid, and cytoskeletal element binding domain analogous to those of protein kinase C. Electron micrograph reconstruction of the nervous system of two unc-13 mutant alleles reveals abnormal gap junctions and chemical synapses between many interneurons, including AVA, AVB, and AVD. Also, there is abnormal axonal morphology of the AS2 motor neuron. Sequence elements of unc-13 indicate that it is a novel component of a signal transduction pathway. Mutant characteristics suggest it is involved in the development of the nervous system and it's function. Our current goal is to resolve the expression pattern of unc-13. Due to the phenotype we believe that it is expressed mainly in neurons, but possibly not in all neurons. The 5' end of the unc-13 mRNA has been identified and used to locate the region containing the unc-13 promoter. The promoter region has been subcloned into a reporter gene construct. Preliminary data using F1 progeny shows staining which appears to be restricted to neurons in the ventral nerve cord, the nerve ring and tail ganglia. In addition, we have generated polyclonal antisera to two portions of the Unc-13 protein. The antisera will be used for western analysis of wild type and unc-13 mutant nematodes and for immunohistochemistry in order to confirm the cellular staining observed with the reporter gene.

Rakow TL et al. (1997) International C. elegans Meeting "CHARACTERIZATION OF THE ELUSIVE UNC-10 GENE"

Schafer WR, Rakow TL

[International C. elegans Meeting, 1997]

In investigating how response to neurotransitters is modulated we have defined a series of mutants that are hypersensitive to serotonin with respect to their egg laying behavior. Mutants of unc-10 fall into this category. In addition to being serotonin hypersensitive, they are aldicarb resistant, indicating defects in acetylcholine synthesis or transmission. Taken together, these two phenotypes lend to our model that acetylcholine plays a role in modulating the response of certain neurons in the egg-laying circuit to serotonin. In addition, unc-10 mutants lay eggs in M9, a phenotype shared with other serotonin hypersensitive mutants. Since the phenotype of unc-10 suggests that it may encode a protein involved in the regulation of neurotransmission, we decided to clone it. In the process of trying to clone unc-10 we have found it necessary to perform mapping experiments to reestablish the map position. It seems that the previous map position assigned to unc-10 is located too far to the right on the X chromosome. Currently, experiments are underway to map unc-10 with respect to nearby STS sites and dpy-7, a gene thought to be relatively close to unc-10. Once the map position has been reassigned, experiments will be initiated to clone unc-10.

Rakow TL et al. (1996) West Coast Worm Meeting "STRUCTURAL STUDIES OF VC NEURONS IN SEROTONIN HYPERSENSITIVE MUTANTS"

Schafer WR, Rakow TL

[West Coast Worm Meeting, 1996]

In the process of uncovering the mechanisms that underlie egg laying in C. elegans, evidence has pointed to a role for negative modulation of serotonin response by another neurotransmitter. Studies in our lab have shown that this modulation is possibly due to acetylcholine, mutants with known defects in acetylcholine synthesis or release are hypersensitive to serotonin and Egl-c. The source of this acetylcholine could be the VC neurons, in particular VC4 and 5 which are "guilty by position", laying on either side of the vulval slit innervating the VM2 muscles. A limited survey of unc's has yielded other mutants that are also serotonin hypersensitive and Egl-c. Two potential reasons for this hypersensitivity are lack of connectivity between the VC's and their target or a defect in the synaptic function of these neurons. To address the possibility of an axonal guidance defect, studies have been done examining the structure of VC4 and 5 using an integrated array which expresses GFP in these cells. Of the mutants examined so far (unc-2, -10, -20, -36 and egl-19), unc-20 has drastic effects in VC cell body mislocalization and axon guidance. The defects are observed in at lest 50% of unc-20 individuals and those animals displaying the phenotye tend to lay earlier eggs than those that appear wild type. The other mutants, three of which are known Ca2+ channel subunits have grossly normal VC morphologies and therefore may be involved in the presynaptic function of the VC's or the reception of their signal by target cells. Studies are underway to use imaging techniques on the VC's to investigate their physiology in wild type and mutant individuals in vivo.

Rakow TL et al. (1996) West Coast Worm Meeting "UNC-13 IS REQUIRED FOR NEUROTRANSMISSION AND LOCALIZED TO PRESYNAPTIC TERMINAL MEMBRANE REGIONS."

Duerr JS, Hosono R, Maruyama H, Rand JB, Eustance V, Maruyama IN, Rakow TL

[West Coast Worm Meeting, 1996]

Mutations in the unc-13 gene cause diverse defects in the nervous system. The mutants have uncoordinated movement, slow irregular pharyngeal pumping, and are resistant to an acetylcholinesterase (AchE) inhibitor, such as aldicarb. They also accumulate abnormally high levels of acetylcholine without altering the level of the precursor choline and choline acetyltransferase activity. It has also been found histochemically that the motor and sensory neurons are misplaced in the mutants. Furthermore, abnormal connections between major interneurons, AVA and AVD, through gap junctions as well as abnormal development of motor neurons have been found by reconstruction of part of the ventral nerve cord, using serial section electron microscopy. Molecular cloning of the unc-13 gene and sequencing of the cDNA reveal that the product encodes a novel protein with a central domain, which has a sequence similar to that of the regulatory region, consisting of the CI and CII subdomains, of the protein kinase C (PKC). It has been demonstrated biochemically that UNC-13 is a high affinity receptor for phorbol esters and diacylglycerol, indistinguishable from PKC in terms of its affinity to the ligands and sensitivity to various PKC inhibitors. Mammalian homologues of unc-13 have also been identified to be expressed exclusively in the brain. These results suggest that UNC-13 involves in a novel signal transduction pathway and plays crucial roles for the development and function of the nervous system. We have further analyzed a role of unc-13 for neurotransmission by making double mutants between unc-13 and other genes including unc-18, ace-2 and ace-3. The unc-18 gene plays a vital role in neurotransmitter release by interacting with syntaxin, consistent with the Unc-18 phenotype of the abnormally high level of acetylcholine accumulation. A double mutant between strong alleles of unc-13 and unc-18 has synthetic lethal phenotype, but a double mutant between their weak alleles is viable and shows a stronger locomotive defect than that of each of the single mutants. This phenotype of the double correlates with a higher level of acetylcholine accumulation than the additive levels of the accumulation in each of the single mutants. The unc-13 ace-2 double mutant has better movement than the unc-13 single mutant. Furthermore, analysis of doubles between unc-13 and ace-3 shows that higher sensitivity of ace-3 against AchE inhibitors such as trichlorfon is suppressed by unc-13 mutations. These results suggest that the unc-13 signal transduction pathway may be parallel to or overlap with neurotransmitter release processes mediated by unc-18. This putative role of UNC-13 is consistent with its molecular structure of the protein, which has three Ca2+ and phospholipid-binding domains similar to synaptotagmin. This hypothesis is further supported by the results that the UNC-13 protein is localized to neuronal processes in a punctate pattern, presumably to synaptic regions. This localization is not altered in neurons of the unc-104 mutant, suggesting that UNC-13 locates on the plasma membrane side, not on the vesicle, in synaptic terminals. Molecular analysis of the unc-13 transcript has revealed that the transcript is processed by alternative trans- and cis-splicing to produce at least three variants. This molecular diversity may explain the diverse defects in the unc-13 nervous system.

Varao AM et al. (2021) J Trace Elem Med Biol "Toxic effects of thallium acetate by acute exposure to the nematode C. elegans."

Varao AM, Ribeiro DE, Onduras A, Gubert P, Silva JDS, Silva LPD, Schwerdtle T, Soares FAA, Alves LC, Amaral LO, Bornhorst J, Filho JLL, Santos CS, Stiboller M, Aleixo LLP

[J Trace Elem Med Biol, 2021]

BACKGROUND: Thallium (Tl) is a toxic metalloid and an emerging pollutant due to electronic devices and dispersal nearby base-metal mining. Therefore, Tl poses a threat to human health and especially the long-term impact on younger individuals exposed is still unknown. This study aimed to evaluate the toxic effects of thallium acetate in C. elegans in early larval stages, considering physiological and behavioral endpoints, as well as the Tl absorption and bioaccumulation. METHODS: concentration to evidence Tl uptake and bioaccumulation. RESULTS: quantification in worms showed its absorption in the L1 larval stage and bioaccumulation in the body after development. CONCLUSIONS: exposure in the early stages of life, even for a short period.

Louzon M et al. (2019) Environ Int "Telomere dynamic in humans and animals: Review and perspectives in environmental toxicology."

Pauget B, Louzon M, de Vaufleury A, Coeurdassier M, Gimbert F

[Environ Int, 2019]

Telomeres (TLs) play major roles in stabilizing the genome and are usually shortened with ageing. The maintenance of TLs is ensured by two mechanisms involving telomerase (TA) enzyme and alternative lengthening telomeres (ALT). TL shortening and/or TA inhibition have been related to health effects on organisms (leading to reduced reproductive lifespan and survival), suggesting that they could be key processes in toxicity mechanisms (at molecular and cellular levels) and relevant as an early warning of exposure and effect of chemicals on human health and animal population dynamics. Consequently, a critical analysis of knowledge about relationships between TL dynamic and environmental pollution is essential to highlight the relevance of TL measurement in environmental toxicology. The first objective of this review is to provide a survey on the basic knowledge about TL structure, roles, maintenance mechanisms and causes of shortening in both vertebrates (including humans) and invertebrates. Overall, TL length decreases with ageing but some unexpected exceptions are reported (e.g., in species with different lifespans, such as the nematode Caenorhabditis elegans or the crustacean Homarus americanus). Inconsistent results reported in various biological groups or even between species of the same genus (e.g., the microcrustacean Daphnia sp.) indicate that the relation usually proposed between TL shortening and a decrease in TA activity cannot be generalized and depends on the species, stage of development or lifespan. Although the scientific literature provides evidence of the effect of ageing on TL shortening, much less information on the relationships between shortening, maintenance of TLs, influence of other endogenous and environmental drivers, including exposure to chemical pollutants, is available, especially in invertebrates. The second objective of this review is to connect knowledge on TL dynamic and exposure to contaminants. Most of the studies published on humans rely on correlative epidemiological approaches and few in vitro experiments. They have shown TL attrition when exposed to contaminants, such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), pesticides and metallic elements (ME). In other vertebrates, the studies we found deals mainly with birds and, overall, report a disturbance of TL dynamic consecutively to exposure to chemicals, including metals and organic compounds. In invertebrates, no data are available and the potential of TL dynamic in environmental risk assessment remains to be explored. On the basis of the main gaps identified some research perspectives (e.g., impact of endogenous and environmental drivers, dose response effects, link between TL length, TA activity, longevity and ageing) are proposed to better understand the potential of TL and TA measurements in humans and animals in environmental toxicology.

Hurtado-Diaz ME et al. (2020) Neurotox Res "Thallium Toxicity in Caenorhabditis elegans: Involvement of the SKN-1 Pathway and Protection by S-Allylcysteine."

Estrada-Valencia R, Verstraeten SV, Santamaria A, Rangel-Lopez E, Hurtado-Diaz ME, Galvan-Arzate S, Karasu C, Maya-Lopez M, Tunez I, Colonnello A, Aschner M

[Neurotox Res, 2020]

Monovalent thallium (Tl⁺) is a cation that can exert complex neurotoxic patterns in the brain by mechanisms that have yet to be completely characterized. To learn more about Tl⁺ toxicity, it is necessary to investigate its major effects in vivo and its ability to trigger specific signaling pathways (such as the antioxidant SKN-1 pathway) in different biological models. Caenorhabditis elegans (C. elegans) is a nematode constituting a simple in vivo biological model with a well-characterized nervous system, and high genetic homology to mammalian systems. In this study, both wild-type (N2) and skn-1 knockout (KO) mutant C. elegans strains subjected to acute and chronic exposures to Tl⁺ [2.5-35M] were evaluated for physiological stress (survival, longevity, and worm size), motor alterations (body bends), and biochemical changes (glutathione S-transferase regulation in a gst-4 fluorescence strain). While survival was affected by Tl⁺ in N2 and skn-1 KO (worms lacking the orthologue of mammalian Nrf2) strains in a similar manner, the longevity was more prominently decreased in the skn-1 KO strain compared with the wild-type strain. Moreover, chronic exposure led to a greater compromise in the longevity in both strains compared with acute exposure. Tl⁺ also induced motor alterations in both skn-1 KO and wild-type strains, as well as changes in worm size in wild-type worms. In addition, preconditioning nematodes with the well-known antioxidant S-allylcysteine (SAC) reversed the Tl⁺-induced decrease in survival in the N2 strain. GST fluorescent expression was also decreased by the metal in the nematode, and recovered by SAC. Our results describe and validate, for the first time, features of the toxic pattern induced by Tl⁺ in an in vivo biological model established with C. elegans, supporting an altered redox component in Tl⁺ toxicity, as previously described in mammal models. We demonstrate that the presence of the orthologous SKN-1 pathway is required for worms in evoking an efficient antioxidant defense. Therefore, the nematode represents an optimal model to reproduce mammalian Tl⁺ toxicity, where toxic mechanisms and novel therapeutic approaches of clinical value may be successfully pursued.

Maruyama H et al. (2001) Neuroscience "Synaptic exocytosis and nervous system development impaired in Caenorhabditis elegans unc-13 mutants."

Rakow TL, Maruyama IN, Maruyama H

[Neuroscience, 2001]

C. elegans mutants defective in unc-13 exhibited severe behavioral abnormalities including paralyzed locomotion and slow pharyngeal pumping and irregular defecation cycle. Consistent with the phenotypes, the mutants accumulated abnormally high levels of the neurotransmitter acetylcholine and were resistant to acetylcholinesterase inhibitors. The unc-13 gene was expressed in most, if not all, neurons when analyzed by using chimeric constructs consisting of the unc-13 promoter and green fluorescence protein or beta -galactosidase reporter gene. While Ca2+-regulated acetylcholine release is lacking, the mutants were still able to release acetylcholine in vivo and in vitro at similar levels to that mediated by the regulated mechanism. Double mutants defective in both unc-13 and other genes involved in synaptic transmission showed the Unc-13 phenotype. rather than other mutant phenotypes, in terms of locomotion as well as of acetylcholine accumulation. Furthermore, electron microscopic reconstruction of the mutant nervous system uncovered that a majority of neurons developed and connected as those in the wild type except for subtle abnormalities including inappropriate connections through gap junctions and morphological alterations of neurons. These results demonstrate that the unc-13 gene product plays an essential role at a late stage in Ca2+-regulated synaptic exocytosis. Neurotransmitters released through the Ca2+-regulated mechanism are required for, but do not play major roles in the nervous system development. The large amount of Ca2+-independent neurotransmitter release observed in the unc-13 mutants suggests that there may be a distinct mechanism from evoked or spontaneous release in neurotransmission.

Vozdek R et al. (2013) Biochim Biophys Acta "Biochemical properties of nematode O-acetylserine(thiol)lyase paralogs imply their distinct roles in hydrogen sulfide homeostasis."

Kozich V, Sera L, Krijt J, Hnizda A, Vozdek R

[Biochim Biophys Acta, 2013]

O-Acetylserine(thiol)lyases (OAS-TLs) play a pivotal role in a sulfur assimilation pathway incorporating sulfide into amino acids in microorganisms and plants, however, these enzymes have not been found in the animal kingdom. Interestingly, the genome of the roundworm Caenorhabditis elegans contains three expressed genes predicted to encode OAS-TL orthologs (cysl-1-cysl-3), and a related pseudogene (cysl-4); these genes play different roles in resistance to hypoxia, hydrogen sulfide and cyanide. To get an insight into the underlying molecular mechanisms we purified the three recombinant worm OAS-TL proteins, and we determined their enzymatic activities, substrate binding affinities, quaternary structures and the conformations of their active site shapes. We show that the nematode OAS-TL orthologs can bind O-acetylserine and catalyze the canonical reaction although this ligand may more likely serve as a competitive inhibitor to natural substrates instead of being a substrate for sulfur assimilation. In addition, we propose that S-sulfocysteine may be a novel endogenous substrate for these proteins. However, we observed that the three OAS-TL proteins are conformationally different and exhibit distinct substrate specificity. Based on the available evidences we propose the following model: CYSL-1 interacts with EGL-9 and activates HIF-1 that upregulates expression of genes detoxifying sulfide and cyanide, the CYSL-2 acts as a cyanoalanine synthase in the cyanide detoxification pathway and simultaneously produces hydrogen sulfide, while the role of CYSL-3 remains unclear although it exhibits sulfhydrylase activity in vitro. All these data indicate that C. elegans OAS-TL paralogs have distinct cellular functions and may play different roles in maintaining hydrogen sulfide homeostasis.

PLOS Pathogens Staff (2014) PLoS Pathog "Correction: Ubiquitin-Mediated Response to Microsporidia and Virus Infection in C. elegans."

PLOS Pathogens Staff

[PLoS Pathog, 2014]

The fourth author's name is spelled incorrectly. The correct name is: Tiffany L. Dunbar. The correct citation is: Bakowski MA, Desjardins CA, Smelkinson MG, Dunbar TL, Lopez-Moyado IF, et al. (2014) Ubiquitin-Mediated Response to Microsporidia and Virus Infection in C. elegans. PLoS Pathog 10(6): e1004200. doi:10.1371/journal.ppat.1004200