Ramirez DS et al. (1996) West Coast Worm Meeting "REGULATION OF EGG-LAYING BEHAVIOR BY ACETYLCHOLINE"

Schafer WR, Ramirez DS

[West Coast Worm Meeting, 1996]

Past work has identified several mutants that are egg-laying constitutive (Egl-c) and serotonin hypersensitive. One of these mutants was found to be cha-1 which is defective in choline acetyltransferase and is both Egl-c and serotonin hypersensitive. These findings suggested to us that acetylcholine (Ach) plays a very important role in regulating sensitivity to serotonin and thus of egg-laying behavior. To investigate this regulation, we tested mutants affecting ACh receptor function for serotonin hypersensitivity (unc-29, unc-38, unc-63, unc-74, and lev-1). Of these Egl-c Unc mutants, unc-29 and lev-1 did not show serotonin hypersensitivity. However, unc-38, unc-63, and unc-74 were identified as causing hypersensitivity to serotonin. The unc-38 gene codes for a protein subunit known to be part of a nicotinic ACh receptor. unc-63 and unc-74 are known to affect the activity of nicotinic ACh receptors, possibly because they help in the assembly of these receptors. These findings make us believe that ACh is working through nicotinic receptors, though we haven't excluded the possibility that ACh may also be working via muscarinic receptors. We will attempt to confirm this belief with further studies. In addition, the focus of our studies has now shifted to include attempting to identify the location of ACh release and the location of the cholinergic receptors, i.e. on nervous vs. muscle tissue or both. A combination of mosaic analysis and studies of cha-1 promoter mutants will be used in an effort to answer these questions.

Ramirez DS et al. (1996) Worm Breeder's Gazette "Acetylcholine is an Important Neurotransmitter for the Regulation of Egg-laying"

Schafer WR, Ramirez DS

[Worm Breeder's Gazette, 1996]

Previously our lab observed that cha-1 mutants, which are defective in choline acetyltransferase, an enzyme required for the synthesis of acetylcholine (Ach), are both Egl-c and serotonin hypersensitive. This suggests that Ach plays an inhibitory role in egg-laying. This is a surprising result since the cholinergic agonist, levamisole, stimulates egg-laying. Further evidence of acetylcholine's regulatory role is that the ace-2; ace-1 double mutant is egg-laying defective (Egl-d). The ace-2; ace-1 double mutant, is defective in two of three Acetylcholine esterases and thus has very low levels of Ach esterase activity. Additionally we have obtained pharmacological evidence for this proposed down-regulatory mechanism. Application of mecamylamine, a general cholinergic antagonist, stimulates egg-laying. Interestingly, carbachol, a general cholinergic agonist also stimulates egg-laying but requires the HSN's while mecamylamine can also stimulate egg-laying in egl-1 mutants. This leads us to the belief that Ach could have a stimulatory role that requires the HSN's and also have an inhibitory role that is parallel to the HSN's. We have tested mutants known to affect Ach receptor function for serotonin hypersensitivity ( unc-29, unc-38, unc-63, unc-74, lev-1, lev-9, and lev-10). All of the mutants conferred hypersensitivity to serotonin except lev-1 and lev-9 gene mutations. These results suggest that Ach is working through nicotinic cholinergic receptors, though we haven't excluded the possibility that Ach may also be working via muscarinic cholinergic receptors. Furthermore, we have attempted to identify the location of Ach release (i.e. from the VA's, VB's, or VC's). Using an unc-17 (e813) allele, shown by Jim Rand to be a promoter mutation which blocks Ach synthesis in the VA's and VB's but not in the VC's, we have found that these mutants are Egl-c and serotonin hypersensitivity. This result is interesting since the VC's, rather than the VA's and VB's, seemed to be more likely to function as regulators of the vulval muscle because of their location and synaptic connections. We are now taking further steps to attempt to identify the location of the cholinergic receptors (i.e. on nervous vs. muscle tissue or both). We are beginning mosaic analysis to answer this question. In addition we are carrying out a screen to identify genes that rescue the Egl phenotype of the ace-2; ace-1 double mutant.

Ramirez DS et al. (1997) International C. elegans Meeting "Regulatory Role of Acetylcholine in Egg-laying Behavior"

Ramirez DS, Schafer WR

[International C. elegans Meeting, 1997]

We are investigating the regulatory role that acetylcholine (Ach) plays in egg-laying behavior. The cholinergic agonist, levamisole, has long been known to stimulate egg-laying. More recently we observed that cha-1 mutants, which are defective in choline acetyltransferase, and thus contain low levels of acetylcholine, are both egg-laying constitutive (Egl-c) and serotonin hypersensitive. This suggests that Ach can act as a negative as well as a positive regulator of egg-laying. Additional evidence that acetylcholine can inhibit egg-laying is that an ace-2; ace-1 double mutant, which has high cholinergic activity due to defects in 2 of 3 cholinesterase genes, is strongly egg-laying defective. We have obtained pharmacological evidence for this proposed down-regulatory mechanism by observing that the application of mecamylamine, a general cholinergic antagonist, stimulates egg-laying. Experiments investigating the time courses for the stimulatory and inhibitory effects of Acetylcholine on egg-laying will be presented. To investigate the molecular mechanism for Ach-dependent modulation of egg-laying, we have also assayed egg-laying behavior in mutants known to affect Ach receptor function. We determined that mutants affecting nicotinic Ach receptors, including unc-29, unc-38, unc-63, unc-74, and lev-10, are egg-laying constitutive and serotonin hypersensitive. Thus Ach appears to act through a nicotinic receptor. We are now attempting to identify the location of the receptors (for example on nervous or muscle tissue) by analyzing unc-29 mosaics.

Zhou T et al. (2010) Mol Biol Evol "Detecting positive and purifying selection at synonymous sites in yeast and worm."

Wilke CO, Zhou T, Gu W

[Mol Biol Evol, 2010]

We present a new computational method to identify positive and purifying selection at synonymous sites in yeast and worm. We define synonymous substitutions that change codons from preferred to unpreferred or vice versa as nonconservative synonymous substitutions and all other substitutions as conservative. Using a maximum-likelihood framework, we then test whether conservative and nonconservative synonymous substitutions occur at equal rates. Our approach replaces the standard rate of synonymous substitutions per synonymous site, dS, with two new rates, the conservative synonymous substitution rate (dS(C)) and the nonconservative synonymous substitution rate (dS(N)). Based on the ratio dS(N)/dS(C), we find that 0.05% of all yeast genes and none of worm genes show evidence of positive selection at synonymous sites (dS(N)/dS(C) > 1). On the other hand, 9.44% of all yeast genes and 5.12% of all worm genes show evidence of significant purifying selection on synonymous sites (dS(N)/dS(C) < 1). We also find that dS(N) correlates strongly with gene expression level, whereas the correlation between expression level and dS(C) is very weak. Thus, dS(N) captures most of the signal of selection for translational accuracy and speed, whereas dS(C) is not strongly influenced by this selection pressure. We suggest that the ratio dN/dS(C) may be more appropriate than the ratio dN/dS to identify positive or purifying selection on amino acids.

Chang H et al. (2019) Adv Exp Med Biol "Pleiotropic Effects of Taurine on Nematode Model for Down Syndrome."

Chang H, Lee DH

[Adv Exp Med Biol, 2019]

Taurine is traditionally used to treat Down Syndrome (DS); however, the actual foundation for this treatment is not well understood. DS patients suffer from disturbance of the proteostasis network (PN) due to aberrant calcium signaling, which eventually causes endoplasmic reticulum stress (ERS). Taurine has been suggested to play a role in modulating calcium homeostasis and ERS. This study examined whether taurine affects DS symptoms using C. elegans- a DS model in which calcineurins, Ca²⁺/calmodulin-dependent protein phosphatase is mutated to null. The DS nematode model has short body length, slow growth, fertility defects, serotonin-resistant egg-laying defects, and faulty thermal sensing. This study focused on whether taurine may ameliorate the severity of DS at the whole-body level, including reduction in ERS. When treated with taurine, DS nematodes appeared to have lower levels of ERS and phenotypes closer to the wild type. DS nematodes also showed improved egg laying efficiency and thermal sensing index comparable to the wild type. Our findings offer a new perspective on the effectiveness of taurine in treating DS and designing therapeutic strategies to lower ERS and restore disrupted PN.

Griffith A M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "C. elegans Model of Down Syndrome"

Griffith A M, Pierce-Shimomura J

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Down syndrome (DS) is the most common genetic cause of mental retardation, occurring at a rate of 1 in 733 live births in the United States. Individuals with DS often suffer from additional neurological and neuromuscular symptoms including congenital heart defects, hypotonia (poor muscle tone), defects in learning and memory, as well as early-onset Alzheimer's disease. While DS is known to result from an extra copy of chromosome 21, it is unknown which specific genes cause the associated phenotypes. The human 21st chromosome encodes 226 known protein-coding genes. Only a small portion of these genes have been studied for potential roles in DS. We have found that, excluding keratin genes, over 85% of the 21st chromosome genes have equivalents in the C. elegans genome suggesting that the worm can be an extremely useful model for DS. We now aim to identify the subset of these genes which may cause neural and/or muscle dysfunction in DS. To this end, we are first focused on identifying the in vivo role of these genes using RNAi. In our initial screen, we found that approximately half of the RNAi treatments caused worms to exhibit abnormal neuromuscular phenotypes. By examining the locomotion, pharyngeal pumping, and neural anatomy of RNAi-treated worms we can further describe how these genes elicit phenotypes related to DS. Furthermore, by generating strains of worms which overexpress genes of interest, we will be able to observe the effects of DS-equivalent doses of the genes and test putative treatments for DS-related phenotypes. Knowledge of the genes which contribute to the phenotypes of DS will help us understand the causes of this condition and aid in the development of novel treatments to improve the quality of life of people with DS.

Busciglio J et al. (2013) Cytogenet Genome Res "Down syndrome: genes, model systems, and progress towards pharmacotherapies and clinical trials for cognitive deficits."

Gardiner KJ, Capone G, O'Byran JP, Busciglio J

[Cytogenet Genome Res, 2013]

Down syndrome (DS) is caused by an extra copy of all or part of the long arm of human chromosome 21 (HSA21). While the complete phenotype is both complex, involving most organs and organ systems, and variable in severity among individuals, intellectual disability (ID) is seen in all people with DS and may have the most significant impact on quality of life. Because the worldwide incidence of DS remains at approximately 1 in 1,000 live births, DS is the most common genetic cause of ID. In recent years, there have been important advances in our understanding of the functions of genes encoded by HSA21 and in the number and utility of in vitro and in vivo systems for modeling DS. Of particular importance, several pharmacological treatments have been shown to rescue learning and memory deficits in one mouse model of DS, the Ts65Dn. Because adult mice were used in the majority of these experiments, there is considerable interest in extending the studies to human clinical trials, and a number of trials have been completed, are in progress or are being planned. A recent conference brought together researchers with a diverse array of expertise and interests to discuss (1) the functions of HSA21 genes with relevance to ID in DS, (2) the utility of model systems including Caenorhabditis elegans, zebrafish and mouse, as well as human neural stem cells and induced pluripotent stems cells, for studies relevant to ID in DS, (3) outcome measures used in pharmacological treatment of mouse models of DS and (4) outcome measures suitable for clinical trials for cognition in adults and children with DS.

Menzel O et al. (2004) Genomics "The Caenorhabditis elegans ortholog of C21orf80, a potential new protein O-fucosyltransferase, is required for normal development."

Reymond A, Antonarakis SE, Mueller F, Vellai T, Takacs-Vellai K, Guipponi M, Menzel O

[Genomics, 2004]

Down syndrome (DS), as a phenotypic result of trisomy 2 1, is the most frequent aneuploidy at birth and the most common known genetic cause of mental retardation. DS is also characterized by other phenotypes affecting many organs, including brain, muscle, heart, limbs, gastrointestinal tract, skeleton, and blood. Any of the human chromosome 21 (Hsa21) genes may contribute to some of the DS phenotypes. To determine which of the Hsa21 genes are involved in DS, the effects of disrupting and overexpressing individual human gene orthologs in model organisms, such as the nematode Caenorhabditis elegans, can be analyzed. Here, we isolated and characterized C21orf80 (human chromosome 21 open reading frame 80), a potential novel protein O-fucosyltransferase gene that encodes three alternatively spliced transcripts. Transient expression of tagged C21orf80 proteins suggests a primary intracellular localization in the Golgi apparatus. To gain insight into the biological role of C21orf80 and its potential role in DS, we isolated its C. elegans ortholog, pad-2, and performed RNA interference (RNAi) and overexpression experiments. pad-2(RNAi) embryos showed failure to undergo normal morphogenesis. Transgenic worms with elevated dosage of pad-2 displayed severe body malformations and abnormal neuronal development. These results show that pad-2 is required for normal development and suggest potential roles for C21orf80 in the pathogenesis of DS.

Ko YJ et al. (2017) Adv Exp Med Biol "Analysis of Taurine's Anti-Down Syndrome Potential in Caenorhabditis elegans."

Lee DH, Ko YJ, Chang H

[Adv Exp Med Biol, 2017]

Down syndrome (DS) patients overexpress human DS critical region gene 1 (hDSCR-1), whose translational product inhibits calcineurin-dependent signaling pathways of genetic transcription. Compared to hDSCR-1, C. elegans rcn-1 has 40% sequence similarity and its proteins share an analogous function with hDSCR-1 in regulating calcineurin. Taurine has had a positive effect on DS patients. According to animal research studies, taurine reduces the expression of MCIP1, a calcineurin inhibitory protein, on C2C12 myotubes and fibroblast in mouse. This study utilizes two C. elegans models for DS: rcn-1 overexpression model, displaying a calcineurin-deficient phenotype, and calcineurin loss-of function mutants. C. elegans larvae were treated with taurine to characterize its effect and mechanism in helping DS patients. RCN-1 expression and behavioral changes were examined in rcn-1 overexpression and calcineurin-deficient models at different concentrations of taurine. When treated with taurine, transgenic worms harboring an rcn-1 reporter (RCN-1::GFP) showed a reduced level of rcn-1 mRNA expression and improved behaviors that were comparable to those in the wild type. These results indicate that taurine exerts a down-regulating effect on the expression of rcn-1 and, consequently, a positive effect on the expression of calcineurins. In summary, taurine may improve the DS symptoms by prompting a positive interaction between RCN-1 and calcineurin. Furthermore, these results suggest that novel mechanisms may regulate interactions among taurine, RCN-1 and calcineurin.

Kieras FJ (1996) East Coast Worm Meeting "Characterization of the Glycosaminoglycans of C. elegans"

Kieras FJ

[East Coast Worm Meeting, 1996]

Glycosaminoglycans(GAGs) were prepared from wild type C. elegans (larvae and adults) by papain digestion of delipidated worms, precipitation with cetylpyridinium chloride(CPC), and further purification by precipitation from high salt solutions with absolute ethanol. The purified GAGs were characterized by TLC and by HPLC analysis of the unsaturated disaccharides produced by chondroitinase digestion. TLC analysis of untreated material revealed a diffuse heterogeneous band with a mobility similar to dermatan sulfate(DS). This was completely degraded by chondroitinase ABC, while treatment with chondroitinase AC I Flavo produced resistant material with less heterogeneity and a mobility similar to DS. HPLC analysis showed the major component to be the 4-sulfated disaccharide. The disulfated disaccharides 4SUA2S and 4S6S which are indicative of DS structure were also present. Therefore the GAGs of wild type C. elegans consist almost entirely of a chondroitin sulfate-dermatan sulfate copolymer.