van der Linden, Alexander et al. (2017) International Worm Meeting "Global accumulation of circRNAs during aging in C. elegans."

Bauer, Matthew, Gruner, Matthew, Voda, Alexandru-Ioan, Miura, Pedro, Gruner, Hannah, Cortes-Lopez, Mariela, van der Linden, Alexander, Cooper, Daphne

[International Worm Meeting, 2017]

Circular RNAs (circRNAs) have recently been identified as a natural occurring family of wide-spread and diverse endogenous non-coding RNAs (reviewed in Cortes-Lopez and Miura, Yale J. Biol, 2016). They are remarkably stable RNA molecules mostly generated by backsplicing events from known protein-coding genes, and are abundantly expressed in many animals, from C. elegans (Ivanov et al, Cell Rep, 2015; Memczak et al, Nature 2013), to humans. Recent reports suggest functional roles for circRNAs, such as regulating transcription, binding proteins, and as microRNA sponges. CircRNAs accumulate during aging on a genome-wide level in Drosophila and mice (Westholm et al, Cell Rep, 2014; Gruner H. et al, Sci Rep, 2016), but their potential role and function in the aging process is not known. We profiled for the first time genome-wide circRNA changes during aging in C. elegans. Using total RNA-seq of whole C. elegans from L4-staged larvae, Day-1, Day-7 and Day-10 adults, we identified 1166 circRNAs including 575 novel annotations. Individual circRNAs were validated by Northern blot, qRT-PCR, and Sanger sequencing. We identified a massive and progressive accumulation of circRNAs during aging, which is independent of linear RNA changes from shared host genes. More specifically, 290 circRNAs were significantly upregulated in Day-10 compared to Day-1 adults, whereas only 6 were downregulated. This includes a >20-fold enrichment in a circRNA derived from the gene for the CREB homolog crh-1, as verified by qRT-PCR. Other genes in longevity-associated pathways produce age-accumulated circRNAs, including daf-16 and daf-2. Unlike in other species, the age-accumulation trend appears not to be confined to neuronally expressed genes. Given the progressive accumulation of circRNAs and their resistance to decay, we propose that stability in post-mitotic tissues is the main driver of the age-upregulation trend. We are currently examining whether circRNA accumulation patterns change in mutants with altered lifespan phenotypes. Taken together, our characterization of age-accumulated circRNAs lays the foundation for future investigations into the functions of circRNAs and how they might play a role in the aging process.

Keith A Boroevich et al. (2004) West Coast Worm Meeting "Identification of Regulatory Elements in Caenorhabditis elegans using Orthology Biased Gibbs Sampling"

Steven JM Jones, David L Baillie, Keith A Boroevich

[West Coast Worm Meeting, 2004]

Much attention has recently been placed on the problem of identification of gene regulatory sequences. Two main computational approaches exist: 1) identification of sequences that share similarity to known regulatory elements, and 2) de novo identification of common motifs in a set of co-regulated genes. Orthology Biased Gibbs Sampling (OrBS) applies a modification of the Gibbs Sampler put forth by Lawrence, Altschult et al . 1 in a attempt to solve this problem. Additional features include analysis of negative strand, multiple (or no) occurrences of a motif in any given sequence and identification of multiple unique motifs, all of which have appeared in previous incarnations of the Gibbs Sampler. The unique feature of OrBS is the use of comparative genomics as an informative prior and in the calculation of motif probability. OrBS will be applied to the identification of regulatory elements C. elegans . Clustering of spatially co-regulated genes as defined by the C. elegans Gene Expression Project (see Johnsen et al . poster). This data is more amenable to regulatory element detection than the more common microarray data because the sequence in which a cis -acting element may reside is strictly defined. Interspecies sequence conservation will be identified through alignment of the C. elegans gene promoter region to the promoter region of the C. briggsae ortholog using LAGAN 2 . Predicted regulatory elements will be verified in vivo using site directed mutagenesis and promoter::GFP fusions. References: 1 Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, Wootton JC. (1993). Science 262: 208-214. 2 Brudno M, Do C, Cooper G, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S. (2003). Genome Research 13: 721-731.

Michelle L. Tucci et al. (2007) International Worm Meeting "Rescue of dopaminergic behavioral deficits induced by alpha-synuclein overexpression via genetic and chemical effectors."

Shusei Hamamichi, Michelle L. Tucci, Cody J. Locke, Guy A. Caldwell, Kim A. Caldwell

[International Worm Meeting, 2007]

Alpha-synuclein is the key molecule involved in synucleinopathies since multiplication of the wildtype alpha-synuclein locus is sufficient for progressive loss of dopamine (DA) neurons and the development of Parkinsons disease. We have constructed two isogenic strains of C. elegans that co-express GFP and alpha-synuclein under the dat-1 promoter and have found that both lines exhibit DA neurodegeneration. Notably, the level of DA neurodegeneration corresponds to the level of alpha-synuclein expression as determined by semi-quantitative RT-PCR. Furthermore, both strains exhibit an age-related decline in DA neuron survival. For example, in worms expressing the higher level of alpha-synuclein, there is almost no degeneration when the animals are 3 days old, while 7 and 10 day-old animals have 25 and 36% degeneration, respectively. In mammalian systems, DA is implicated in the neurotoxicity of alpha-synuclein in DA neurons. We have discovered that the dorsal CEP neurons, which are post-synaptic to the ADE dopamine-producing neurons, degenerate significantly more often than the ventral CEP neurons, which do not synapse onto other dopamine producing neurons (p=0.02; Fisher Exact Test). DA neurodegeneration induced by alpha-synuclein overexpression was significantly rescued by overexpression of several genes implicated in Parkinsons disease (Cao et. al., J Neuroscience 25:3801; Cooper et al., Science 313:324; Hamamichi et al., 16th International Worm Meeting). Furthermore, several chemicals isolated from ongoing drug screening efforts significantly enhance DA neuron survival in our model. Alpha-synuclein-expressing worms were also tested for deficits in the DA-specific behavior of basal slowing in response to the presence of bacteria (Sawin et al., Neuron 26:619). We found that these worms displayed a locomotory defect for this phenotype that was often observed prior to the loss of DA neurons. Thus, it is possible that the behavioral deficit represents a more sensitive readout for DA neuron loss than visual inspection of the neurons. Furthermore, genetic and chemical enhancers of DA neuron survival often rescued this DA-specific behavioral phenotype.

Shu Hamamichi et al. (2007) International Worm Meeting "Hypothesis-based RNAi Screening to Identify Neuroprotective Genes in a Parkinson's Disease Model."

Kim A. Caldwell, Guy A. Caldwell, Adam L. Knight, Renee N. Rivas, Shu Hamamichi

[International Worm Meeting, 2007]

Genomic multiplication of the locus encoding human alpha-synuclein, a polypeptide with a propensity toward intracellular misfolding, results in Parkinson''s disease (PD). Using worms overexpressing human alpha-synuclein::GFP fusion in body wall muscles, we have established conditions that facilitate the rapid identification of factors that influence the misfolding of this protein over time. Here we report the results of a systematic RNAi screen of approximately 950 candidate genetic targets to identify factors that enhance misfolding of alpha-synuclein over the course of aging in C. elegans. The mature bioinformatics data associated with C. elegans biology provides an opportunity to more rapidly discern novel candidate effector proteins for subsequent functional analysis. In this context, our targets were primarily prioritized based on hypothetical bioinformatic associations to existing genes (parkin, Nurr-1, UCHL-1, DJ-1, PINK-1, LRRK2, and ATP13A2) and cellular pathways (ubiquitin-proteasome system, ER-associated degradation pathway, unfolded protein response, and autophagy) with implications for PD. This screen revealed 14 genes that when knocked-down, consistently resulted in increased alpha-synuclein misfolding; none of these genes were associated with a prior link to PD. We previously established the application of C. elegans toward functional validation of an evolutionarily conserved and neuroprotective protein, Rab1, using a neurodegenerative model of age- and dose-dependent alpha-synuclein-induced degeneration in the dopamine neurons of transgenic worms (Cooper et al., 2006, Science). Here we significantly expand our list of neuroprotective genes through subsequent analysis of the positive targets from our RNAi screen. We generated transgenic nematodes that overexpressed the cDNAs corresponding to a prioritized subset of these hits in dopamine neurons. Strikingly, these animals revealed that 5 out of 7 tested gene products significantly protect against dopamine neuron loss. These proteins include two trafficking proteins, two proteins of unknown function with high homology in humans, and one gene already reported to cause neurodegeneration in knockout mice. Our combined bioinformatic/genomic screening approach provides a valuable paradigm for the investigation of disease mechanism using C. elegans. Furthermore, the gene products identified from these functional analyses represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting 1% of the population over 65 years of age.

Suzan J Holt et al. (2003) International Worm Meeting "A dauer approach to survival: Modulate internal oxygen exposure and manufacture metabolites de novo."

Donald L Riddle, Suzan J Holt

[International Worm Meeting, 2003]

A lowered metabolic rate and availability of stored lipids are thought to contribute to the longevity of dauer larvae, which can remain non-feeding for months. At least 5% external oxygen is required for lipid utilization in starving Caenorhabditis (1). The small size of a C. elegans dauer larva, 20 m in diameter, might indicate that oxygen passage by cuticular diffusion is sufficient for maintenance of aerobic physiology. Nevertheless, recent evidence from gene expression profiles suggests that anaerobic metabolism is augmented in dauer larvae. Serial analysis of gene expression (SAGE) has revealed that the dauer larva, though developmentally arrested, has a surprisingly complex transcriptome (2). We compared transcript abundance between the dauer and mixed-stage profiles for 59 genes with confirmed or predicted involvement in carbohydrate metabolism. The most prominent transcript was for phosphoenolpyruvate carboxykinase (PEPCK). The pyruvate kinase:PEPCK SAGE tag ratio for either profile supports a glycolytic shunt such as that found in parasitic helminths which are characterized by succinate fermentation and anaerobic ATP formation. However, the transcript patterns for key enzymes in the glyoxylate cycle, gluconeogenesis, and for glycogen phosphorylase reflected higher potential involvement for PEPCK in carbohydrate synthesis in mixed stages compared to the dauer. Transcripts for additional proteins with predicted functions in oxygen-independent routes for energy generation were dauer-enriched. The morphology of dauer larvae together with apparent upregulation of anaerobic pathways indicates that the dauer interior could be hypoxic. These data may help explain the hypoxia resistance (Hyp) of N2 dauer larvae and may have implications regarding the adult Hyp phenotype of certain daf-2 mutants (3). The lowering of the aerobic metabolic rate, as well as oxygen level, may lower the rate of oxidative damage accumulation and contribute to dauer longevity, as long as the -oxidation of lipids is not seriously compromised. In addition, fermentation byproducts, used as alternative nutrient sources, could serve to slow the depletion of lipid stores and extend the survival period.1. Cooper and Van Gundy. 1970. J. Nematol. 2:305; 2. Jones, Riddle, Pouzyrev, Velculescu, Hillier, Eddy, Stricklin, Baillie, Waterston, and Marra. 2001. Genome Res. 11:1346; 3. Scott, Avidan, and Crowder. 2002. Science 296:2388.

Alex Holmes et al. (2006) European Worm Meeting "DAB-1 Gives Us New Insights into Trafficking Pathways"

Jonathan Pettitt., Alex Holmes

[European Worm Meeting, 2006]

Alex Holmes & Jonathan Pettitt. The mammalian Disabled proteins are cargo-specific adaptor proteins that facilitate the endocytosis of lipoprotein receptors. We, and others, have obtained data implicating the sole C. elegans Disabled, DAB-1, in the trafficking of the lipoprotein receptors LRP-1/-2 (Kamikura & Cooper, 2003; Holmes & Pettitt, IWM, 2005), suggesting that this is a conserved function of Disabled proteins. We have shown that DAB-1 is expressed in oocytes, early embryos, and in all epithelia of late stage embryos and postembryonic stages. It forms membrane-associated puncta that may correspond to clathrin-coated pits or vesicles. DAB-1 puncta are largely confined to the apical membranes of cuticle-secreting epithelia, such as the hypodermis and pharynx: in contrast they are enriched on the basolateral membranes of intestinal cells. Strong punctate expression is also seen on the surfaces of coelomocytes, consistent with the coelomocyte uptake (Cup) phenotype observed in dab-1 null mutants. DAB-1::GFP expression during endocytosis in epithelial tissues and coelomocytes is particularly dynamic, and we are currently investigating whether DAB-1 puncta dynamics in the hypodermis are subject to developmental regulation, such as during moulting. We are also examining the role of clathrin and the AP-2 complex on puncta turnover. We have previously identified a synergistic genetic interaction between AP-1-mediated trafficking and DAB-1 mediated endocytosis. Similarly, loss-of-function mutations affecting components of the AP-3 complex are synthetic lethal in combination with dab-1 mutations. This suggests that DAB-1 is a component of a pathway that acts in parallel to AP-1 and AP-3 to traffic molecules from the plasma membrane. Thus, in the absence of AP-1 or AP-3 dependent pathways, some cargoes normally sorted by these adaptors can still reach their normal destinations via DAB-1-dependent endocytosis from the plasma membrane.. We want to identify receptors participating in DAB-1-dependent endocytosis. Coelomocytes endocytose a broad range of substrates suggesting that the receptor(s) involved can bind to a wide range of ligands. Lipoprotein receptors, such as megalin, are known to recognise a broad range of ligands making these good candidates for endocytic receptors acting in coelomocytes. We have identified 16 transmembrane proteins containing the DAB-1-binding motif, [F/Y]xNPxY, in their intracellular domain. Five of these are members of the lipoprotein receptor family: LRP-1, LRP-2, RME-2, T13C2.6 and APL-1. It is likely that not all identified receptors bind only to DAB-1, since NUM-1 and CED-6 have also been shown to bind to NPxY motifs. We are carrying out systematic RNAi knockdown of each of these genes to determine whether this will phenocopy the dab-1 null phenotype. Combined RNAi of lrp-1 and lrp-2 results in a high level of embryonic/larval lethality with a greatly increased frequency of moulting defects, indicating that these molecules have overlapping functions, however coelomocyte endocytosis is not noticeably defective.

Swan KA et al. (1996) West Coast Worm Meeting "CYTOKINESIS IN C. ELEGANS EMBRYOS REQUIRES THE MATERNAL GENE cyk-1."

Swan KA, Martin PR, Bowerman BA

[West Coast Worm Meeting, 1996]

We have identified a recessive, maternal-effect, embryonic-lethal mutation in a C. elegans gene we call cyk-l, for cytokinesis-defective. Mothers homozygous for the cyk-1(or36) mutation produce embryos that undergo a largely normal first cell cycle and initiate cytokinesis during the first nuclear mitosis. A contractile ring and a cleavage furrow form at the beginning of cytokinesis, but eventually the cleavage furrow regresses, leaving the two daughter nuclei in a single cell. The mutant embryos continue to undergo multiple rounds of nuclear mitosis, during which time cytokinesis continues to initiate but almost invariably fails to be completed, resulting in the development of one-cell embryos filled with many nuclei that even differentiate (as detected by staining fixed mutant embryos with cell-type specific antibodies). Two other processes that resemble cytokinesis are also defective: the mutant embryos fail to extrude polar bodies during meiosis and exhibit reduced furrow formation during the pseudocleavage process that occurs while the maternal and paternal pronuclei migrate towards each other during the first cell cycle. However, other processes in the early embryo that require a functional cytoskeleton appear to occur normally. Pronuclear migration and rotation of the first mitotic spindle are unaffected, the first mitotic spindle is displaced posteriorly, and P-granules and PAR-1 protein are localized to the posterior cortex during the first cell cycle, as in wild-type. Thus the requirement for cyk-l appears highly specific for cytokinesis and related processes. The cyk-1(or36) mutation maps to about position -1.0 on LGIII. This sequenced region includes a predicted gene similar to genes known to be required for cytokinesis in other organisms, including diaphanous in D. melanogaster, cdc-12 in S. pombe, and bni-1/she-5 in S. cerevisiae (thanks to Steve Wasserman for first pointing out this similarity). Germ-line transformation showed that the cosmid containing this predicted gene rescues cyk-1 mutant embryos. Anti-sense RNA corresponding to one exon of the diaphanous-related gene, when injected into the germ-line of wild-type mothers, results in an embryonic cytokinesis defect indistinguishable from that caused by or36. We conclude that cyk-1 represents a newly identified member of this gene family, members of which are implicated not only in cytokinesis but also in the establishment of cell polarity (bni-1/she-5 and cappuccino in D. melanogaster). Because cytokinesis appears to initiate normally in cyk-1 mutant embryos, we are interested in the possibility. that cyk-l function is required for a late step in cytokinesis, perhaps even in termination. Late events in cytokinesis may in turn be important for the formation of remnant bodies and CAS structures, capping protein and actin containing structures known to be involved in orienting spindle axes during early embryonic cleavages in C. elegans (Hyman and White, 1987; Waddle, Cooper, and Waterston, 1994).