Snarrenberg, C. et al. (2013) International Worm Meeting "Expanding the Spectrum of Dopamine Regulators: Swip-10, the (m)BLAC Sheep of the Family."

Snarrenberg, C., Whitaker, S., Han, Q., Blakely, R.D., Pohl, E., Hardaway, J.A.

[International Worm Meeting, 2013]

Several neurological disorders are associated with disrupted dopamine (DA) signaling including Parkinson's disease, schizophrenia and addiction. In C. elegans, an inability to clear synaptic DA results in swimming induced paralysis (Swip). In a screen for animals that display DA-dependent Swip, we identified two mutants that support DA-dependent Swip, vt29 and vt33, and that lie within a previously undescribed gene F53B1.6, now named swip-10. Sequence analysis revealed highly conserved mouse and human orthologs, Mblac1 and MBLAC1 respectively, named for the presence of metallo-b lactamase domains. Swip-10 promoter:GFP fusion constructs yielded expression in C. elegans glial cells in vivo. Consistent with this localization, rescue of Swip was achieved by expressing swip-10 under either its own promoter or the ptr-10 glial promoter. In the mammalian brain, glial cells regulate DA neurons and DA-modulated circuits by clearing extracellular glutamate via expression of high affinity glutamate transporters. Immunocytochemical studies reveal high levels of Mblac1 expression in the mouse brain in areas associated with glutamatergic control of emotional and reward behaviors including the prefrontal cortex, hippocampus, nucleus accumbens, habenula and amygdala. In keeping with a role of swip-10 in regulating glutamate-dependent DA neuron excitation, we found that mutation of multiple GLTs generates DA-dependent Swip and that swip-10 and glt-4 interact genetically to trigger Swip. Additionally, loss of the glutamate receptors, glr-4, glr-6 and mgl-1, individually and in combination diminished the Swip of swip-10. Our results suggest that swip-10 is a novel regulator of extracellular glutamate via altered glutamate transport. We hypothesize that knockout of swip-10 elevates tonic extracellular glutamate, increases excitation of DA neurons, and increases DA release. Further analysis of swip-10 and its mammalian orthologs may lead to novel insights into brain disorders linked to altered glutamate signaling, including those for which perturbed glutamate-control of DA signaling has been documented. F31MH09312 to JAH and R01MH095044 to RDB.

Refai, O. et al. (2019) International Worm Meeting "Disruption of Mitochondrial Function Supports Dopamine-Dependent Paralysis and Neurodegeneration in C. elegans."

Miller, G.W., Mellish, S., Kalia, V., Blakely, R.D., Hardaway, J.A., Rodriguez, P., Refai, O.

[International Worm Meeting, 2019]

Dopamine (DA) is a neurotransmitter that modulates neural signaling across phylogeny. In humans, disruption of DA signaling is associated with multiple brain disorders including ADHD, schizophrenia, addiction and Parkinson's disease. The DA transporter (DAT) regulates the spatial and temporal dynamics of extracellular DA following vesicular release. Human DAT has been shown to be able to rescue phenotypes associated with a loss of function mutation (lof) in the C. elegans DAT ortholog, dat-1, supporting a functional conservation of the transporter in regulating DA signaling. Genetic and pharmacological elimination of DAT-1 function results Swimming-induced paralysis (Swip), a phenotype that we have used previously to identify and characterize novel regulators of DA signaling. Here, we present our efforts to characterize one of these mutants, swip-20. Drugs that suppress DA signaling in humans, reserpine and azaperone, as well as genetic mutations that inhibits DA secretion and signaling were found to restore normal swimming behavior of swip-20 animals, supporting an impact of swip-20 mutation on DA signaling. Moreover, overexpression of DAT-1 also restored swimming behavior of swip-20 animals. Using SNP mapping and whole genome sequencing, the molecular lesion incurred by mutagenesis of swip-20 was mapped to a locus in chromosome III, as a missense mutation in the catalytic site of a highly-conserved protein bearing a short-chain dehydrogenase/reductase domain. Prior studies across phyla indicate that the swip-20 gene encodes a protein required for efficient mitochondrial respiration and RNA processing, that in humans has been linked to neurodegenerative disease. In support of our gene identification, energetic and metabolomic analyses revealed swip-20 mutants to display significantly reduced mitochondrial respiration, elevated oxidized glutathione, and a disrupted redox state, relative to wild-type animals. The in vivo oxidative stress reporter Pgst-4::GFP exhibited elevated expression in swip-20 mutants. Neurodegeneration and morphological defects were also observed in DA neurons of Swip-20 mutants, suggesting that paralysis derives from poorly controlled DA release and/or hyper-functional postsynaptic DA receptors. Ongoing studies linking disrupted glutamate signaling as a contributor to Swip and neurodegeneration will be presented. Together, our studies establish a new C. elegans model for the study of energetic loss-driven deficits in motor function and neuronal viability.

Tauffenberger A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Glucose rescues age-dependent proteotoxicity in C.elegans"

Vaccaro A, Parker A, Tauffenberger A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

As the population shifts to an older demographic, it has become important to find new therapeutic ways to combat neurological disease. To model neurodegeneration we use a C. elegans model for Huntington's Disease (HD). HD is a late-onset fatal neurodegenerative disease caused by an expanded CAG trinucleotide repeat that codes for glutamine (Q), and is characterized by the intracellular accumulation of mutant proteins. Our transgenic C. elegans strains expressing normal (19Q) and expanded mutant (128Q) polyQ in mechanosensory neurons show a progressive and polyQ size dependent loss-of-mechanosensation and increased-aggregation phenotypes in vivo1. The difference between mutant (128Q) and controls (19Q) is easily distinguished and the effects of genetic and pharmacological modifiers can be rapidly derived from small populations of animals2. Disrupted metabolism is one of a number of mechanisms that may contribute to neurodegeneration. Indeed, conditions that reprogram metabolism like dietary restriction are active areas of investigation in efforts to identify new therapeutic targets. We have observed that opposite to the effects on organismal lifespan, excess glucose suppresses toxicity in worm models of proteotoxicity. Furthermore, many of the genes essential for glucose-mediated suppression of proteotoxicity are in common with genes required for dietary restriction. These data demonstrate that glucose enrichment and dietary restriction utilize many of the same genetic pathways with opposite metabolic endpoints both of which are beneficial in models of neurodegeneration. A summary of our recent data will be presented. 1.Parker, J.A. et al. Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. Proc Natl Acad Sci U S A 98, 13318-23 (2001). 2.Parker, J.A. et al. Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons. Nat Genet 37, 349-50 (2005).

Giannis Voglis et al. (2005) International Worm Meeting "Specific DEG/ENaC ion channels are required for proper learning and memory in C. elegans"

Nektarios Tavernarakis, Giannis Voglis

[International Worm Meeting, 2005]

Members of the DEG/ENaCs superfamily of ion channels have been identified in nematodes, snails, flies and vertebrates including humans, where they serve diverse functions such as ion homeostasis and sensory transduction. Members of the acid-sensing subgroup of DEG/ENaC ion channels (termed ASICs) have been implicated in synaptic plasticity, learning and memory in mammals (Wemmie J.A. et al., Neuron, 34: 463-77). In C. elegans, the DEG/ENaC protein family comprises 30 members, expressed in a variety of cell types, from neurons to muscles and epithelia. Seven have been characterized genetically and are implicated in mechanosensation, proprioception, and in the regulation of ultradian rhythms. There is very little information on the physiological role of the rest 23 members, and their possible involvement in signal transduction. We find that ASIC-1, a novel DEG/ENaC protein and close homologue of mammalian ASIC, is highly expressed in head sensory neurons and interneurons. ASIC-1 is required for conditioning to several chemical attractants, while it is dispensable for chemotaxis. Consistently, these genes are also required for associating the presence of food with the rearing temperature. Such observations indicate that DEG/ENaCs contribute to the capacity of the animal for associative learning. Therefore, ASIC roles in associative learning and memory might be conserved from nematodes to mammals, thus, making C. elegans an attractive model in which to dissect the relevant mechanisms.

G Voglis et al. (2004) European Worm Meeting "SPECIFIC DEG/ENAC ION CHANNELS ARE REQUIRED FOR PROPER LEARNING AND MEMORY IN C. ELEGANS"

G Voglis, D Bazopoulou, N Tavernarakis

[European Worm Meeting, 2004]

Members of the DEG/ENaCs superfamily of ion channels have been identified in nematodes, snails, flies and vertebrates including humans, where they serve diverse functions such as ion homeostasis, sensory transduction and memory. In C. elegans, the DEG/ENaC protein family comprises 30 members, expressed in a variety of cell types, from neurons to muscles and epithelia. Seven have been characterized genetically and are implicated in mechanosensation, proprioception, and in the regulation of ultradian rhythms. There is no information on the physiological role of the rest 23 members, and their possible involvement in signal transduction. We have undertaken a comprehensive study of uncharacterized DEG/ENaC genes in C. elegans aiming to elucidate their function. To this end we have determined the spatiotemporal expression and are generating knock-out alleles for all genes. Our analysis has revealed that specific DEG/ENaC proteins, which are highly expressed in head sensory neurons and interneurons, are required for conditioning to several chemical attractants, while they are dispensable for chemotaxis. Consistently, these genes are also required for associating the presence of food with the rearing temperature. Such observations indicate that DEG/ENaCs contribute to the capacity of the animal for associative learning. Interestingly, similar findings in mice support the involvement of the related, acid-sensing ion channel (ASIC) in synaptic plasticity, learning, and memory (Wemmie J.A. et al., Neuron, 34: 463-77). Therefore, DEG/ENaC roles in associative learning and memory might be conserved from nematodes to mammals, thus, rendering C. elegans an attractive model in which to dissect the relevant mechanisms.

Cheryl Van Buskirk et al. (2004) West Coast Worm Meeting "A physiological role for let-23/EGFR in C. elegans growth"

Paul Sternberg, Cheryl Van Buskirk

[West Coast Worm Meeting, 2004]

The single EGF receptor homolog in C. elegans, LET-23, has multiple functions during development. LET-23 signals through the ras/MAPK pathway to specify cell fates in the ventral cord, vulva, male tail, and excretory system(1). LET-23 also signals through a PLC/IP3 pathway to regulate ovulation(2). We have observed that overexpression of the EGFR ligand LIN-3 from a heat shock inducible promoter, in addition to affecting the expected cell fates, also slows down growth of the animals as compared to heat-shocked controls. This growth defect, which affects time to adulthood but not the final size of the animal, occurs in response to overexpression of LIN-3 at any developmental stage, and thus is unlikely to be due to a cell fate specification defect. The growth defect is suppressible by mutations in let-23, and we are currently testing known downstream targets of LET-23 for suppression to determine which pathway is involved. We are also investigating possible underlying causes of this growth phenotype and we will present these data along with our genetic analysis of this previously undescribed physiological role for let-23 signaling. 1. Moghal, N.M. and P.W. Sternberg. 2003. The Epidermal Growth Factor System in Caenorhabditis elegans. Exp. Cell Res. 284, 150-159. 2. Clandinin, T.R, DeModena, J.A., and P.W. Sternberg. 1998. Inositol Trisphosphate mediates a RAS-Independent Response to LET-23 Receptor Tyrosine Kinase Activiation in C. elegans. Cell 92, 523-533.

Giannis Voglis et al. (2007) International Worm Meeting "An acid sensing ion channel mediates associative learning in C. elegans by modulating dopamine signaling."

Giannis Voglis, Nektarios Tavernarakis

[International Worm Meeting, 2007]

We have conducted a systematic characterization of putative acid-sensing ion channels (ASICs) in C. elegans. ASICs are members of the DEG/ENaC family of ion channels and have been identified in diverse organisms, serving different functions such as ion homeostasis and sensory transduction. Mammalian ASICs have been implicated in synaptic plasticity, learning and memory (Wemmie J.A. et al., Neuron, 34: 463-77).We find that ASIC-1, a novel C. elegans acid-sensing ion channel and a close homologue of mammalian ASICs is required for associative learning in the nematode. ASIC-1 is localized at presynaptic terminals of dopaminergic neurons. We find that animals lacking ASIC-1 are defective in associative learning although they show normal sensory perception. Exogenous addition of dopamine or elimination of the DAT-1 dopamine transporter counterbalance the defects associated with ASIC-1 deficiency. Similarly, genetic ablation of neurons expressing asic-1 impairs associative learning, which is restored by exogenous addition of dopamine. Removal of the dopamine receptors DOP-1 and DOP-2, but not DOP-3, recapitulates the effects of asic-1 deletion on associative learning. Furthermore, synaptic release at dopaminergic neurons is compromised in the absence of ASIC-1 activity. Thus, ASIC-1 functions in dopaminergic neurons to mediate normal dopaminergic signaling, necessary for associative learning in C. elegans. Our results reveal a novel role of DEG/ENaC ion channels in modulating neuronal communication by regulating the activity of dopaminergic synapses. Similar mechanisms may facilitate synaptic plasticity in vertebrates.

Fassnacht, N. et al. (2019) International Worm Meeting "Chromatin remodeling mediates repair of DNA damage in C. elegans germ cells."

Peruffo , A., Fassnacht, N., Checchi, P.

[International Worm Meeting, 2019]

Chromatin remodeling proteins are required for multiple aspects of DNA repair. This is particularly important in germ cells, wherein defective repair affects gamete quality. This in turn leads to infertility, miscarriage, and chromosomal disorders. Previously, we discovered a role for the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex in the repair of programmed DNA lesions. Loss of the catalytic component of NuRD, known as the Mi2 complex, disrupts repair of meiotic DSBs1. Here we investigated the role of the Mi2 component LET-418 in response to several forms of exogenous DNA damage. We assessed embryonic viability in response to several mutagens including nitrogen mustard and cisplatin, which cause inter- and/or intra-strand crosslinks (ICLs), a byproduct of replication errors. Our data reveal reduced embryonic viability in a let-418 loss-of-function mutant when mitotically dividing germ cells are exposed to these agents. This is consistent with previous findings that Mi2 has a role in the repair of double-stranded breaks, which can be formed from ICLs. We are currently assessing the molecular nature as to how LET-418 responds to ICLs and other types of DNA lesions, such as those caused by hydroxyurea and UVC. Overall, our findings support a role of Mi2 in maintaining genomic stability in multiple contexts. 1Turcotte, C.A., Sloat, S.A., Rigothi, J.A, Rosenkranse, E., Northrup, A., Andrews, N.P., and Checchi, P.M. (2018). Maintenance of genome integrity by Mi2 homologs CHD-3 and LET-418 in Caenorhabditis elegans. Genetics. 208:1-17.

Stephan Steidl et al. (2002) West Coast Worm Meeting "Mutations of a glutamate-gated chloride channel in C. elegans affect short-term memory in an ISI dependant manner, long-term memory, and foraging behaviour"

Nicholas Dube, Catharine H Rankin, Stephan Steidl, Jacqueline K Rose

[West Coast Worm Meeting, 2002]

avr-14 encodes a alpha-type subunit of a glutamate-gated chloride channel (GluCls) in C. elegans, a receptor closely related to mammalian glycine-gated chloride channels. avr-14 is expressed on the glutamatergic sensory neurons of the tap-withdrawal response (TWR) (Dent et al., 2000). avr-14 receptors may act as an autoreceptors regulating sensory neuron activity. Worms with a functional null mutation of avr-14 were tested for short-term habituation of the TWR at 10s, 30s, 45s, and 60 s ISI's. It was found that worms with this mutation showed more rapid habituation than wild-type at shorter ISIs (10s and 30s) while showing habituation similar to wild-type at long ISIs (45s and 60s). Worms with the same mutation appear to show enhanced long-term memory compared to wild-type controls. avr-14 mutants show an increased frequency and a decreased magnitude of spontaneous reversals compared to wild-type. Spontaneous reversals are thought to be part of a foraging strategy in C. elegans. The hypothesis that the altered frequency and magnitude of spontaneous reversal would affect foraging strategy was supported by an experiment is which avr-14 worms were unable to negotiate a simple copper sulfate maze to reach food while wild-type worms easily reached the food. Reference: Dent, J.A., Smith, M.M., Vassilatis, D.K., & Avery, L. (2000). The genetics of Ivermectin resistance in Caenorhabditis elegans. Proc. Natl. Acad. Sci. (USA), 97, 2674-2679. Supported by: An NSERC PGS A to SS and an NSERC operating grant to CHR.

Kami Montgomery et al. (2001) International C. elegans Meeting "A Structure/Function Analysis of the sys-1 Gene"

Jennifer Miskowski, Katie Donlevy, Judith Kimble, Kami Montgomery, Ron Ellis

[International C. elegans Meeting, 2001]

We are interested in understanding how a complex organ of the correct size, shape, and function is generated. To address this, we are studying formation of the C. elegans gonad. We have identified the sys-1 gene that is required for hermaphrodite gonad development, but is not essential for male gonad development. Hermaphrodites that are homozygous for sys-1(q544) , a putative null allele, exhibit two interesting defects. First, they lack distal tip cells, and the cells that normally make distal tip cells can generate extra anchor cells (Miskowski et al., 2001). This phenotype is similar to that described for mutants in the Frizzled homolog, lin-17 . (Sternberg and Horvitz, 1988). Second, they fail to form the somatic gonadal primordium during late L2, which rearranges cells into a prepattern of the adult organ. To gain insight into the mechanism by which sys-1 acts, we have pursued cloning of the sys-1 gene. The sys-1 locus was mapped near fog-3 on LGI by three-factor mapping and deficiency analysis. In that region, a gene was identified that rescues the sys-1 mutant phenotype and mimics the sys-1(q544) defect by RNAi. This gene encodes a novel, cytoplasmic protein. A putative C. briggsae homolog of this gene has been identified; it is over 50% identical to its C. elegans counterpart at the amino acid level. We are currently investigating the function of the C. briggsae gene by RNAi. Using the sequence similarity between C. briggsae and C. elegans , we are deleting the most conserved regions from the C. elegans gene and testing the activity of these reconstructed genes by transformation rescue. We hope that this analysis will help elucidate the function of this novel protein. Miskowski, J.A. et al. (2001) Dev Biol 230: 61-73. Sternberg, P.W. and Horvitz, H.R. (1988) Dev Biol 130: 67-73.