Toshiaki Suzuki et al. (2003) International Worm Meeting "Analysis of Ce-PARKIN in C. elegans"

Nobutaka Hattori, Toshiaki Suzuki, Keiji Tanaka, Yoshikuni Mizuno, Shohei MItani, Noriyuki Matsuda, Hideki Yashiroda, Toshiaki Kitami, Keiko Gengyo-Ando

[International Worm Meeting, 2003]

Autosomal recessive juvenile parkinsonism (AR-JP) is one of the most common forms of familial parkinsons disease characterized by selective loss of dopaminergic neurons in substantia nigra and the locus coeruleus. parkin is the causative gene of AR-JP. The human parkin gene encodes 465 amino acids with a ubiquitin-like domain in the amino-terminus and two RING finger motifs in the carboxy terminus. So far, various deletion mutations and point mutations have been discovered in patients of AR-JP, suggesting that the loss of function of Parkin is the cause of AR-JP. Recently we and others showed that Parkin has a ubiquitin-protein ligase activity and therefore suggested that the defect of protein degradation in the neurons of AR-JP patients (Shimura H. et al. Nat. Genet. 25, 302-5, 2000). To investigate the function of Parkin in vivo, we began to analyze the Ce-PARKIN of C. elegans. Two deletion mutations in parkin genes show no defect in their viabilities. The expression of Ce-PARKIN seems to be specific to subset of neurons. Therefore, we focused on the function of Ce-PARKIN in the neurons and the analysis is underway.

Yoshida M et al. (2001) Biochem Biophys Res Commun "Important role of junctophilin in nematode motor function."

Yoshida M, Sugimoto A, Takeshima H, Ohshima Y

[Biochem Biophys Res Commun, 2001]

Junctional complexes between the plasma membrane and endoplasmic/sareoplasmic reticulum are shared by excitable cells and seem to be the structural ground for cross-talk between cell-surface and intracellular ionic channels. Our current studies have identified junctophilins (JPs) as members of a novel transmembrane protein family in the junctional membrane complex. Biochemical and gene-knockout studies have suggested that JPs contribute to the formation of the junctional membrane complex by spanning the intracellular store membrane and interacting with the plasma membrane. We report here invertebrate JPs in fruit fly and nematode. Three distinct JP subtype genes are found in the mammalian genome, while a single JP gene exists in either invertebrate genome. Mammalian and invertebrate JPs share characteristic structural features, although some intervening sequences are found in invertebrate JPs. A reporter assay indicated that the JP gene is predominantly activated in muscle cells in nematode. Nematodes, in which expression of JP was inhibited by RNA-mediated interference (RNAi), showed hypolocomotion. Taking account of the cell-type-specific expression and data from previous reports, the hypolocomotion is likely to be due to the deficiency of junctional membrane structures and the resulting reduction of Ca2+ signaling during excitation-contraction coupling in muscle cells.

Park J et al. (2006) Nature "Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin."

Kim J, Park J, Bae E, Shong M, Kim JM, Lee SB, Kim Y, Song S, Lee S, Kim S, Chung J

[Nature, 2006]

Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset form of Parkinson''s disease characterized by motor disturbances and dopaminergic neurodegeneration. To address its underlying molecular pathogenesis, we generated and characterized loss-of-function mutants of Drosophila PTEN-induced putative kinase 1 (PINK1), a novel AR-JP-linked gene. Here, we show that PINK1 mutants exhibit indirect flight muscle and dopaminergic neuronal degeneration accompanied by locomotive defects. Furthermore, transmission electron microscopy analysis and a rescue experiment with Drosophila Bcl-2 demonstrated that mitochondrial dysfunction accounts for the degenerative changes in all phenotypes of PINK1 mutants. Notably, we also found that PINK1 mutants share marked phenotypic similarities with parkin mutants. Transgenic expression of Parkin markedly ameliorated all PINK1 loss-of-function phenotypes, but not vice versa, suggesting that Parkin functions downstream of PINK1. Taken together, our genetic evidence clearly establishes that Parkin and PINK1 act in a common pathway in maintaining mitochondrial integrity and function in both muscles and dopaminergic neurons.

(1990) Worm Breeder's Gazette "The Contig Database at NIG Japan"

[Worm Breeder's Gazette, 1990]

A node for the C. elegans genome map database is open at NIG ( National Institute of Genetics) Japan. It can be accessed by two ways as below: (1) Via telephone line: The number is 0559-75-6036, 6037 (8bit no parity, 1200,2400 bps) (2) Telnet access via Internet: The node name is DDBJ.NIG.AC. JP For the users whose computers are not linked to Internet, special accounts (loginname: a88657 or a88657a, password: DDBJ-NIG) on the computers of five university computer centres (Tokyo University, Nagoya University, Kyoto University, Osaka University, Kyushu University) are available to have Telnet access to the NIG computer. Once you login to the account, your terminal is automatically connected to the NIG computer (charge free!). In both cases, the login name is C_ELEGANS and no password is necessary. If you have any questions, feel free to contact Yuji Kohara at 0559-75-0771, ext. #693 or ykohara@niguts.nig.ac. jp

Springer W et al. (2005) Hum Mol Genet "A Caenorhabditis elegans Parkin mutant with altered solubility couples alpha-synuclein aggregation to proteotoxic stress."

Springer W, Hoppe T, Schmidt E, Baumeister R

[Hum Mol Genet, 2005]

Mutations in the human parkin gene encoding an E3 ubiquitin ligase have been associated with early-onset recessive forms of Parkinson''s disease (PD). However, the molecular mechanisms by which mutations in the parkin gene cause PD are still under debate. Here, we identified and characterized the Caenorhabditis elegans parkin homolog, pdr-1. PDR-1 protein physically associates and cooperates with a conserved degradation machinery to mediate ubiquitin conjugation. Strikingly, in contrast to pdr-1 loss-of-function mutants, an in-frame deletion variant with altered solubility and intracellular localization properties is hypersensitive towards different proteotoxic stress conditions. Both endoplasmic reticulum-derived folding stress and cytosolic stress conferred by expression of mutant human alpha-synuclein resulted in severe developmental defects and lethality in pdr-1(lg103) mutant background. Furthermore, we show that the corresponding truncated protein PDR-1(Deltaaa24-247) aggregates in cell culture, but still interacts with its ubiquitylation co-enzymes. Thus, it might block the cellular degradation/detoxification machinery and therefore renders worms highly vulnerable to protein folding stress. In contrast to other complete gene knock-outs or RNAi models of Parkin function, this C. elegans model recapitulates Parkin insolubility and aggregation similar to several AR-JP linked Parkin mutations. We suggest that such Parkin variants that either confer a neomorphic function or a partial loss-of-function may help to further elucidate the biological function of Parkin in vivo and the pathogenic mechanisms resulting in AR-JP. Due to high-throughput capacity of C. elegans, this model is particularly well suited to identify genetic and chemical modifiers of toxicity.

Elling AA et al. (2007) Genome Biol "Divergent evolution of arrested development in the dauer stage of Caenorhabditis elegans and the infective stage of Heterodera glycines."

Mitreva M, Baum TJ, Martin J, Gai X, McK Bird D, McCarter JP, McDermott JP, Davis EL, Nettleton D, Elling AA, Maier TR, Recknor J, Hussey RS, Hewezi T

[Genome Biol, 2007]

ABSTRACT: BACKGROUND: The soybean cyst nematode Heterodera glycines is the most important parasite in soybean production worldwide. A comprehensive analysis of large-scale gene expression changes throughout the development of plant-parasitic nematodes has been lacking to date. RESULTS: We report an extensive genomic analysis of Heterodera glycines, beginning with the generation of 20,100 expressed sequence tags (EST). In-depth analysis of these EST plus approximately 1,900 previously published sequences predicted 6,860 unique H. glycines genes and allowed a classification by function using InterProScan. Expression profiling of all 6,860 genes throughout the H. glycines life cycle was undertaken using the Affymetrix Soybean Genome Array GeneChip. Our data sets and results represent a comprehensive resource for molecular studies of H. glycines. Demonstrating the power of this resource, we were able to address whether arrested development in the Caenorhabditis elegans dauer larva and the H. glycines infective second-stage juvenile (J2) exhibits shared gene expression profiles. We determined that the gene expression profiles associated with the C. elegans dauer pathway are not uniformly conserved in H. glycines and that the expression profiles of genes for metabolic enzymes of C. elegans dauer larvae and H. glycines infective J2 are dissimilar. CONCLUSIONS: Our results indicate that hallmark gene expression patterns and metabolism features are not shared in the developmentally arrested life stages of C. elegans and H. glycines, suggesting that developmental arrest in these two nematode species has undergone more divergent evolution than previously thought and pointing to the need for detailed genomic analyses of individual parasite species.

P Polk et al. (1999) International C. elegans Meeting "In Search of Phenotypes for ptl-1"

P Polk, E Aamodt, S Aamodt

[International C. elegans Meeting, 1999]

In mammals, the microtubule-associated protein tau is located in the axons of neuronal cells and is believed to stabilize the microtubule cytoskeleton and function in neurite outgrowth. A C. elegans homologue of tau, ptl-1 (protein with tau-like repeats), was identified by the C. elegans Genome Sequencing Consortium and characterized by us (McDermott et al, 1996, Biochemistry 35: 9415-9423) and Goedert et al. (1996, J. Cell Sci. 109:2661-2672). We obtained a C. elegans strain with a deletion of the repeat region from the NemaPharm Group at Axys Pharmaceuticals. The ptl-1 deletion strain has no apparent phenotype and we have not detected differences between N2 and the ptl-1 deletion strain in sensitivity to colchicine, benomyl or cytochalasin D. Work done by N. Hirokawa and colleagues has shown that in mice, tau functions redundantly with microtubule-associated protein MAP1B. Mice homozygous for a null allele of tau (Harada et al., 1994, Nature 369: 488-491) or MAP-1B (Takei et al., 1997, J. Cell Biol. 137: 1615-1626) have mild phenotypes, but double mutants have severe defects in brain development as a result of defects in neurite outgrowth (Takei et al., 1998, Mol. Biol. of the Cell 9:394a). Because of this precedent for redundancy in the function of tau and MAP-1B, we searched for C. elegans genome for a MAP1B homologue but no candidate was found. To attempt to identify proteins that act cooperatively or are redundant to ptl-1 , we have begun a screen for synthetic mutations. We crossed the ptl-1 deletion into a strain (obtained from David Baillie) that has cosmid F42G9 (containing ptl-1 ) and rol-6 as an extrachromosomal array. We will mutagenize the ptl-1 deletion strain containing the F42G9 array to screen for genes that are synthetic to ptl-1 .

Aamna Kaul et al. (2003) International Worm Meeting "Hyperactivation of glutamate-gated chloride channels with ivermectin results in necrosis."

Joseph Dent, Aamna Kaul

[International Worm Meeting, 2003]

Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991

Andersen*, Kristine et al. (2015) International Worm Meeting "An Investigation into the Affect of Neuronal Activity on Proper Neural Connectivity in C. elegans."

Andersen*, Kristine, Varshney, Aruna, Duong, Alex, Miller, Kristine, Park, Joori, Pyle, Jacqueline, Barsi-Rhyne*, Benjamin, VanHoven, Miri, Vargas, Christopher, Holdrich, Emma, Tsujimoto, Bryan, Tran, Alan

[International Worm Meeting, 2015]

Neuronal activity has been implicated in the establishment and maintenance of appropriate synaptic connections in vertebrate and invertebrate systems. However, the molecular mechanisms by which neuronal activity affects connectivity are poorly understood. To understand this process, we have focused our studies on the PHB phasmid sensory neurons. We have begun to elucidate the pathway by which sodium dodecyl sulfate (SDS) is sensed by the phasmid neurons using a high throughput assay adapted from the method developed by Hilliard and colleagues (Current Biology, 2002). Briefly, SDS is sensed by the PHB neurons, which terminate backward movement via their connections with AVA interneurons. Using this assay, we find that odr-3/Galphaolf and tax-2/CNG-channel beta subunit, in addition to previously discovered tax-4/CNG-channel alpha subunit (Hilliard et al., Current Biology, 2002), are required for SDS chemosensation. To determine if defects in sensory signaling affect sensory synapses, we utilized the split-GFP-based trans-synaptic marker NLG-1 GRASP (Neuroligin-1 GFP Reconstitution Across Synaptic Partners) to visualize synapses between PHBs and AVA interneurons in live animals. Interestingly, we find that neuronal activity is required for maintaining these sensory synapses. Time course experiments indicate that odr-3/Galphaolf mutant L1s have normal synapses, but synapses are significantly reduced in later larval stages, although phasmid neuron morphology appears to be unaffected. Cell-specific rescue experiments indicate that odr-3/Galphaolf likely functions in PHB neurons. Subcellular localization of odr-3/Galphaolf shows localization to the PHB cilia, consistent with a role in sensory signaling being required to maintain synaptic integrity. These results indicate that C. elegans may be a powerful model organism for elucidating the molecular mechanisms by which sensory activity affects synaptic connectivity. Our future goal is to further characterize the mechanism by which sensory activity maintains synapses using molecular genetic and physiological approaches. Funded by NIH (1R01NS087544 to MV at SJSU and NL at UCSF, 5T34GM008253 MARC undergraduate fellowship to CV, 2R25GM071381 RISE undergraduate fellowships to CV and JP), HHMI (SCRIBE 52006312 undergraduate fellowship to BB and KM), and NSF (RUMBA REU 1004350 fellowship to KA and EH).

Wolfdieter Springer et al. (2003) International Worm Meeting "A C. elegans model for Parkinsons Disease"

Ralf Baumeister, Wolfdieter Springer, Thorsten Hoppe

[International Worm Meeting, 2003]

Parkinsons Disease (PD) is the second most common neurodegenerative disorder, affecting about two percent of the population over the age of 65. It is a chronic, progressive disease pathologically characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Most cases of PD are sporadic, but some familial cases of PD have so far been linked to mutations in a few different genes. Mutations in the parkin gene cause autosomal recessive juvenile Parkinsonism (AR-JP), an inherited form of PD with very early onset. We identified and characterized the C. elegans orthologue of human parkin, which we named pdr-1 (Parkinsons Disease related). PDR-1 shows the same architectural structure, with 28 % identity and 41 % similarity on protein level. Parkin/PDR-1 is a RING-containing E3 ubiquitin ligase required for ubiquitin-proteasome dependent degradation of specific protein substrates. The ubiquitin-proteasome system is the major non-lysosomal proteolytic pathway in all eukaryotes, which regulates turnover of short-lived and misfolded proteins in the cytosol and the nucleus. Misfolded proteins in the endoplasmatic reticulum (ER) are retrotranslocated across the ER membrane into the cytosol and subsequently get degraded by the ER-associated degradation pathway (ERAD). Increased formation of misfolded proteins in the ER lumen leads to the activation of the unfolded protein response (UPR). This pathway results in a translational attenuation and a transcriptional upregulation of genes facilitating protein folding and degradation of misfolded proteins. We show conserved function of PDR-1 by using yeast interaction studies and GST-pulldown experiments characterizing protein interactions and enzymatic activity. We will present data showing that it interacts with ubiquitin-conjugating enzymes (E2) and with the C. elegans orthologue of CHIP. This multiubiquitin chain assembly factor (E4) is involved in Parkin-dependent ubiquitination of its substrate. Furthermore we established an in vitro ubiquitination assay demonstrating the self-ubiquitination abililty of PDR-1, which is characteristic for RING-containing E3 ligases. To determine the in vivo function we constructed a deletion mutant of pdr-1. At first, even extensive phenotypic analyses did not reveal an obvious defect in the pdr-1 deficient animals. However, using a combination of genetic and pharmacological methods, we identified a crucial role of PDR-1 in the ERAD pathway during UPR. Thereby we present a powerful in vivo system to study PD on the cellular and molecular level.