Hanna Farah et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Preferential Interactions of RIC-3 with nAChR Subunits"

Millet Treinin, Hagit Cohen Ben-Ami, Hanna Farah

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

In C. elegans RIC-3 is required for the maturation of multiple nicotinic acetylcholine receptors, including the DEG-3/DES-2 neuronal nAChR. RIC-3 belongs to a conserved gene family. Members of this family all encode two transmembrane domains followed by one or two coiled-coil domains. Analysis of the effects of RIC-3 on the DEG-3/DES-2 receptor in Xenopus leavis oocytes shows affects on both receptor peak current amplitudes and on receptor properties. RIC-3's effects on DEG-3/DES-2 properties are similar to the effects of increasing the DEG-3 to DES-2 subunit ratio, suggesting that RIC-3 may preferentially enhance maturation of DEG-3 rich receptors. All effects of RIC-3 in Xenopus oocytes are mediated by a short sequence encompassing the transmembrane domains but lacking the coiled-coil domains. We now show that effects on both receptor properties and receptor peak amplitudes are reduced by mutation of a single conserved residue in the second transmembrane domain. In addition we show that RIC-3 interacts with DEG-3 in the absence of DES-2 but not with DES-2 in the absence of DEG-3. Last we show that RIC-3 enhances surface expression of DEG-3 but not of DES-2. Thus our analysis shows a linkage between the different effects of RIC-3 on the DEG-3/DES-2 receptor, and shows that effects on receptor properties can be explained by a preferential interaction of RIC-3 with the DEG-3 subunit, leading to increased surface expression of DEG-3 rich receptors.

Lina Yassin et al. (2003) International Worm Meeting "DEG-3 function and dysfunction"

Lina Yassin, Millet Treinin

[International Worm Meeting, 2003]

DEG-3 is a subunit of a nicotinic acetylcholine receptor (nAChR) that can mutate to cause neuronal degeneration. In Xenopus oocytes DEG-3 together with DES-2, another nAChR subunit, form a channel. Analysis of this channel led to a model whereby physiological concentrations of choline, an agonist of the DEG-3/DES-2 channel, lead to constitutive activity of the mutant channel, leading to excessive entry of calcium and cell death. Analysis of the DEG-3/DES-2 channel in oocytes provided basic insights into the causes of neuronal degeneration. But the analysis also implied differences between DEG-3/DES-2 channel activity in oocytes and in native neurons. To understand these differences we added to the oocyte system factors likely to affect DEG-3 channel activity in C. elegans. Two such factors, co-expression with RIC-3 and chronic exposure to choline were found to modify DEG-3/DES-2 channel activity. Choline is expected to exert this effect in-vivo because it is a necessary metabolite constitutively found in most organisms. RIC-3 is required for maturation of multiple nAChRs (including DEG-3) in C. elegans and enhances nAChR surface expression in oocytes. Interestingly manipulations that modify DEG-3/DES-2 channel stoichiometry mimic the effects of RIC-3 on channel properties, suggesting a second role for RIC-3 in regulating subunit stoichiometry. Taken together the identification of factors that modify DEG-3/DES-2 channel activity improves our understanding of its normal and pathological activity, and suggests that in-vivo these factors and others, yet to be identified, regulate receptor numbers and properties.

Yassin L et al. (2000) European Worm Meeting "Characterization of the DEG-3/DES-2 receptor: an early branch of the nicotinic acetylcholine receptor family"

Kahan T, Halevi S, Yassin L, Gillo B, Eshel M, Treinin M

[European Worm Meeting, 2000]

The nicotinic acetylcholine receptor family (nAChR) is a large family of acetylcholine gated cation channels. The great diversity of this family as seen in most multi-cellular organisms suggests that this family emerged early in evolution. We are characterizing the DEG-3/DES-2 nAChR. Subunits of this receptor, DEG-3 and DES-2, where first identified due to their involvement in neuronal degeneration. Both genes belong to an early branch of the nAChR family tree. Pharmacological analysis of a DEG-3/DES-2 receptor expressed in Xenopus oocytes shows that this receptor, like the a-7 nAChR, is activated by choline. However, unlike a-7, choline is the preferred agonist of the DEG-3/DES-2 receptor. We also show that in C. elegans the DEG-3/DES-2 receptor is localized to non-synaptic regions, including the sensory endings of chemosensory neurons. This localization is in agreement with a role for this receptor in chemosensation of choline, as inferred from a defect in chemotaxis for choline seen in deg-3 mutants. Our results combined with phylogenetic analysis of the nAChRs suggest a non-synaptic origin for these receptors, and a role in chemotaxis for the ancestors of this family.

Treinin M et al. (1998) European Worm Meeting "CHARACTERIZATION OF GENES INVOLVED IN NEURONAL DEGENERATION"

Eshel M, Yassin L, Treinin M

[European Worm Meeting, 1998]

deg-3 is a subunit of a nicotinic AChR that can mutate to cause neuronal degeneration, a process typified by extensive cell swelling followed by cell death (Treinin and Chalfie, (1995). Neuron 14: 871-877). Studies of deg-3 induced cell degeneration and the morphologically similar degenerin induced degenerations, suggested a model linking channel hyperactivity to cell death. This model suggests that cell swelling is the result of increased ion influx to the cell, and the concomitant osmotic imbalance. In order to characterize the deg-3 dependent degeneration process we have screened for suppressors of this process. This screen led to the identification of 8 genes (11 mutations), suggesting a complex network of genes interacting in the degeneration process. Initial characterization of these mutations includes analyzing their effect on the degeneration process, as measured by the number of swollen cells, and by the number of surviving cells. This analysis shows that these mutations belong to two groups: 1. Supppressors which have no effect on cell swelling but do result in increased cell survival. The existence of this type of suppressors suggests that cell swelling does not lead directly to cell death. 2. Suppressors which have a strong suppressing effect on both cell swelling and cell death. This suppression of cell swelling (seen in mutations affecting des-2 and des-5), suggests a reduction in mutant DEG-3 channel activity. Indeed, des-2 codes for a nAChR subunit (formerly named deg-3b, or acr-4) which is part of the deg-3 operon. Expression of this gene and deg-3 in an heterologous expression system, Xenopus oocytes, shows that both are needed for channel formation (Gillo, et al. International Worm Meeting 1997, p.109). Thus des-2 codes for a subunit of the DEG-3 channel. des-5, the second gene of this group, appears to play a role in regulating the activity of many C. elegans nAChRs, as mutations in this gene also lead to levamisole resistance, and reduced pharyngeal pumping.

LA Herndon et al. (1999) International C. elegans Meeting "Mutations That Suppress Degenerative Cell Death Can Extend Lifespan"

M Driscoll, LA Herndon

[International C. elegans Meeting, 1999]

Several genetic "conditions" including mec-4(u231), deg-3(u662) and activated G a S induce swelling and degeneration of neurons. This death occurs independently of the programmed cell death pathway. mec-4(u231) [the example we study most] specifies a hyperactive channel subunit and may kill cells via a mechanism similar to mammalian excitotoxic cell death. To learn more about the degenerative death mechanism, we screened for mutations that block mec-4(d) -induced death. An integrated array of p unc-8 mec-4(u231) causes swelling and degeneration of some interneurons and many ventral cord neurons, resulting in severe paralysis. Using this behavioral phenotype, we screened for suppressor mutations that block the deleterious effects of mec-4(u231) : suppressors restore normal or near normal locomotion by preventing cell death. The genes identified by these mutations are called des genes (degeneration suppressor). In a screen of 45,000 haploid genomes, we isolated 30 independent death suppressors. 14 strong suppressors appear to affect novel genes. Three of the strongest suppressors on chromosome V, defined by alleles bz29, bz30 and bz31 , are particularly interesting because they suppress degeneration induced by multiple insults. We have identified a candidate rescuing cosmid for bz29 and bz30 , and are in the process of cloning this gene. Aging nematodes accumulate vacuolar structures that look similar to mec-4(d) -induced necrosis. This led us to wonder whether animals in which degenerative cell death is blocked by mutation might age differently. We hypothesized that if degenerative cell death plays a critical role in C. elegans aging, and if des mutations suppress aging-associated vacuolation, our des mutants might have extended lifespans. Indeed, both bz29 and bz30 (alleles of the same gene) have an extended lifespan, similar to what is seen in age-1 (hx546) animals. At least one more des allele, affecting a different gene, has an extended lifespan. We are testing the long-lived lines to determine the extent of vacuole formation during aging, general locomotor activity and resistance to heat and oxidative stress. In addition, we are determining whether there is an interaction between the des mutations and other known mutations that affect lifespan.

Jun-ichi Aikawa (2001) International C. elegans Meeting "Molecular characterization of heparan sulfate/heparin N-deacetylase/N-sulfotransferase in worms"

Jun-ichi Aikawa

[International C. elegans Meeting, 2001]

Heparan sulfate binds and activates a large variety of growth factors, enzymes and extracellular matrix proteins. These interactions largely depend on the specific arrangement of sulfated moieties and uronic acid epimers within the chains. These oligosaccharide sequences are generated in a step-wise manner, initiated by the formation of a linkage tetrasaccharide which is then extended by copolymerization of alternating a1,4GlcNAc and b1,4GlcA residues. As the chains polymerize, they undergo a series of sulfation and epimerization reactions. The first set of modifications involves the removal of acetyl units from subsets of GlcNAc residues, and the addition of sulfate groups to the resulting free amino groups. These reactions are catalyzed by a family of enzymes designated as GlcNAc N-deacetylase/N-sulfotransferases (NDST), since they simultaneously. Four members of the family have been identified in vertebrates, with single orthologs present in Drosophila and C. elegans. We have revealed tissue-specific expression pattern and unique enzymatic properties of these four isozymes1,2). In fly, loss of NDST (sulfateless) results in unsulfated chains and defective signaling by multiple growth factors and morphogens. I reconstituted cDNA for worm NDST from EST clones and 5' RACE products. Enzymatic activities will be discussed. 1) Aikawa, J. & Esko, J. D J. Biol. Chem. 274, 2690-2695 (1999) 2) Aikawa, J., Grobe, K., Tsujimoto, M. & Esko, J. D J. Biol. Chem. 276, 5876-5882 (2001)

L Yassin et al. (1999) International C. elegans Meeting "Characterization of the DES-2/DEG-3 receptor, an ancient member of the nAChR family"

L Yassin, B Gillo, M Eshel, M Treinin

[International C. elegans Meeting, 1999]

DEG-3 is a subunit of a nicotinic acetylcholine receptor (nAChR) that can mutate to cause neuronal degeneration. deg-3 is part of an operon containing a second nAChR subunit, des-2 . DES-2 is necessary for DEG-3 dependent channel formation in Xenopus oocytes, and for deg-3(u662) induced degenerations. Thus the two genes probably interact in forming a channel in-vivo (Treinin et al, PNAS 95:15492). Sequence analysis of both deg-3 and des-2 show that they are highly divergent members of the nAChR family. Both show similar low (30%) identity to different branches of the nAChR family, suggesting that they branched early from the family tree. Indeed, electrophysiological analysis of the DES-2/DEG-3 receptor expressed in Xenopus oocytes shows that it is a highly divergent nAChR, as application of choline leads to much higher responses than the application of acetylcholine (ACh). Choline was already shown to be an agonist of a -7, also a nAChR, but ours is the first demonstration of a receptor which is more sensitive to choline then to ACh. Choline being a metabolite is unlikely to play a role in synaptic transmission. Accordingly, DEG-3 is distributed all over the cell (as visualized by antibody staining), showing no preference to synaptic regions. As part of the analysis of this divergent nAChR we are also doing structure function analysis, using mutations in deg-3 which where identified in screens for suppressors of deg-3(u662) induced degeneration. Sequences of the mutations demonstrate structural conservation within the nAChR family, as all missense mutations affect conserved residues. Genetic analysis of the mutations has identified a small number of negative dominant suppressors. These mutations are likely to effect functional attributes of the receptors and will be further analyzed using electrophysiology.

Husson, Steven J. et al. (2011) International Worm Meeting "C. elegans mutants defective in neuropeptide amidation enzymes are abnormal in egg-laying behavior."

Landuyt, Bart, Schoofs, Liliane, Gottschalk, Alexander, Horvitz, H.Robert, Temmerman, Liesbet, Husson, Steven J., Ringstad, Niels, Meelkop, Ellen

[International Worm Meeting, 2011]

Egg laying has mainly been studied at the behavioral, neuronal and neurochemical levels, but little is known about the biochemical control of the relevant neuropeptidergic signaling systems. Biosynthesis of endogenous peptides requires processing enzymes, such as proprotein convertase 2, which is encoded by egl-3 (1, 2), and a carboxypeptidase encoded by egl-21 (3, 4). Mutants defective in these genes have egg-laying defects, consistent with the finding that FMRFamide-like peptides (FLPs) have been linked to egg laying behavior. C. elegans enzymes that carry out the last step in the production of biologically active peptides, the carboxy-terminal amidation reaction, have not been characterized. This multistep reaction involves hydroxylation of the glycine a-carbon by a peptidyl-a-hydroxylating monooxygenase (PHM), followed by a cleavage reaction performed by peptidyl a-hydroxyglycine a-amidating lyase (PAL) to generate a glyoxylate molecule and the a-amidated peptide. In vertebrates, both enzymatic activities responsible for the carboxyterminal amidation reaction are contained in one bifunctional enzyme, peptidylglycine a-amidating monooxygenase (PAM). By contrast, invertebrates generally express two separate enzymes encoded by two different genes. Here we report the identification and characterization of C. elegans amidating enzymes using bioinformatics to identify candidate genes and mass spectrometry to compare the neuropeptides in wild-type and newly generated mutants. Mutants lacking a functional PHM displayed an altered neuropeptide profile, showed impaired egg laying behavior and had a decreased brood size. Interestingly, PHM mutants still displayed fully processed amidated neuropeptides, probably as a result of the presence of a bifunctional PAM, the main amidating enzyme in vertebrates. Our data indicate the existence of a robust complementation system for the amidation reaction of neuropeptides in nematodes and suggest the involvement of amidated neuropeptides in egg laying. (1) S. J. Husson et al., J. Neurochem. 98, 1999 (2006); (2) J. Kass et al., J. Neurosci. 21, 9265 (2001); (3) S. J. Husson et al., J. Neurochem. 102, 246 (2007); (4) T. C. Jacob, J. M. Kaplan, J. Neurosci. 23, 2122 (2003).

Herndon LA et al. (1998) East Coast Worm Meeting "A suppressor of degenerative cell death extends lifespan in C. elegans"

Herndon LA, Xu K, Driscoll MA

[East Coast Worm Meeting, 1998]

Nematodes exhibit visible changes in behavior and appearance over the course of their lives. Aging worms feed, move and defecate more slowly than their younger counterparts and have an appearance that is rough and lumpy with a generally distorted morphology. The bodies of aging animals contain vacuoles and cellular inclusions reminiscent of those that occur during degenerative cell death. Cell death is a highly regulated process that plays important roles in normal development and in response to cellular injury. Our lab focuses on the mechanisms of injury-induced cell death, also known as necrosis. Necrotic cell death is characterized by cellular swelling, distention of subcellular organelles and chromatin clumping. In aging cells, damage to cellular DNA, proteins and organelles accumulates and contributes to their dysfunction. Whether injury-induced cell death contributes in a critical way to the aging of an organism, however, remains an unanswered question. That vacuolar necrotic-like cells have often been observed in aging nematodes is an intriguing finding that indicates a possible relationship between necrosis and senescent decline. Therefore, we have extended our analysis of necrotic cell death mechanisms to address the potential influence of degenerative cell death on aging in vivo. To identify genes required for degenerative cell death in C. elegans, we screened for mutations that block necrosis. The genes identified by these mutations are called des genes (degeneration suppressors). Two des genes, identified by alleles bz29 and bz30 in one case and bz31 in a second case, are particularly interesting because they can suppress degeneration induced by several distinct cellular insults and are thus most likely to act in the death process rather than specifically in the regulation of a degenerin channel. We hypothesized that if these mutations also suppress aging-associated vacuolation and if degenerative cell death plays a critical role in C. elegans aging, the des mutants might have extended lifespans or exhibit increased vitality late into their lifespans. To address whether degenerative cell death might contribute to nematode lifespan or senescence, we are characterizing random vacuole formation, general locomotor activity, and lifespan extension in des mutants. We show that animals mutant in des bz29 and bz30 have an extended lifespan, similar to what is seen in age-1(hx546) animals. These studies provide the first link between cell death and aging in C. elegans.

Crittenden SL et al. (1997) International C. elegans Meeting "TEMPERATURE-SENSITIVE REPRESSION OF TRANSGENES CARRYING A fem-3(gf) MUTANT 3'UTR"

Crittenden SL, Kimble JE

[International C. elegans Meeting, 1997]

We are developing a method to control gene expression in a temperature dependent manner. Our strategy is to regulate reporter expression with a well-defined promoter at the 5' end and a mutant fem-3 3'UTR at the 3' end. The fem-3(gf) mutations map to the fem-3 3'UTR and release fem-3 from post-transcriptional repression at 25 C, but not at 15 C (1,2). We have tested expression of a lag-2 promoter::GFP construct that carries either a strong or a weak fem-3(gf) 3'UTR. The lag-2 promoter drives expression in the distal tip cells of adults (3-5). We find that transgenes express GFP at much lower levels at 15 C than at 25 C when carrying a fem-3(gf) 3'UTR. We are currently trying to optimize repression at 15 C using the lag-2 promoter and GFP as a reporter. In addition, we are testing whether temperature sensitive repression can be achieved in other tissues, and we are starting to explore the biological activity of various proteins expressed in the distal tip cell. 1. Ahringer, J., and Kimble, J. (1991). Nature 349, 346-348. 2. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Genetics 115, 107-119. 3. Fitzgerald, K., and Greenwald, I. (1995). Development 121, 4275-4282. 4. Gao, D. and J. Kimble, midwest worm meeting 1996. 5. Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924.