Shen, Linjing et al. (2019) International Worm Meeting "Role of Tar DNA binding protein-43 (TDP-43) in mediating alpha-synuclein (HASN) toxicity in a Parkinson's disease model."

Shen, Linjing, Wang, Changliang, Leung, Ka Lai, Wong, Garry, Chen, Liang

[International Worm Meeting, 2019]

Parkinson's disease (PD) is a progressive neurodegenerative disease mainly affecting the motor system. To date, the treatment for PD remains symptomatic, and the molecular mechanisms underlying its pathophysiology are still unclear. ?-Synuclein is the major component of protein aggregates in Lewy bodies and Lewy neurites of PD. Mutation or increased expression of ?-synuclein likely contributes to the development of PD. Treatment against the ?-synuclein aggregation might be an efficient therapeutic strategy . TAR DNA binding protein-43(TDP-43), mainly responsible for the pathology of amyotrophic lateral sclerosis, is also involved in PD. The synucleinopathy of PD is more serious when ?-synuclein proteinopathy and TDP-43 proteinopathy coexist, but the effects of interaction between ?-synuclein and TDP-43 in PD remain unclear. This study investigates the effect of TDP-43 on ?-synuclein. Our study found that the deletion of the gene tdp-1, TDP-43 ortholog in Caenorhabditis elegans (C. elegans), caused behavior defects to improve and human ?-synuclein (HASN) total protein and aggregation to decrease in the HASN overexpressing C. elegans PD model. These effects may be related to upgraded expression of genes belonging to the heat shock protein family, hsp-70, hsp-16.1, hsp-16.11, hsp-16.41, hsp-16.48, hsp-16.4, hsp-16.2, which were identified through both mRNA sequencing and RNA interference. These studies will further our knowledge on the interactions between TDP-43 and ?-synuclein and their roles in PD. The genes from the heat shock protein family might also be considered as promising therapeutic targets for PD and other ?-synuclein related diseases. This research was supported in part by the Research grant of Garry Wong MYRG2017-00123-FHS.

Huang, Xiaobing et al. (2019) International Worm Meeting "A transgenic co-expressed Amyloid-beta and alpha-Synuclein dementia model in Caenorhabditis elegans."

Huang, Xiaobing, Leung, Ka Lai, Wang, Changliang, Chen, Liang, Shen, Linjing, Wong, Garry

[International Worm Meeting, 2019]

Purpose: Dementia with Lewy bodies (DLB) is the second most common type of progressive dementia after Alzheimer's disease dementia. Lewy bodies develop in nerve cells in brain regions where thinking, memory and movement are lost. Still, the pathology of DLB remains vague. For exploring the pathogenesis of DLB, we constructed a disease model of DLB which co-expressed Amyloid-? (A?) and ?-synuclein in pan-neurons in C. elegans. Methods: We used the locomotion, thrashing, lifespan, development and egg laying behavior assays to evaluate whether co-expressed A? and ?-synuclein would alter phenotypes in worms. Furthermore, we used confocal microscopy to analyze the neurodegeneration of worms and Thioflavin-S staining to observe the misfolded protein aggregation. Moreover, we used RNA-Seq to explore the effects of co-expressed A? and ?-synuclein in worms on transcription. Results: Our results showed that co-expressed A? and ?-synuclein severely impaired the behaviors and affected neurons in C. elegans. Worms that co-expressed A? and ?-synuclein showed uncoordinated and slower movement, shorter lifespan, less progeny and egg laying defects. Moreover, misfolded protein aggregation and damage of dopaminergic neurons were found in co-expressed A? and ?-synuclein worms, suggesting that co-expression of these 2 transgenes can cause damage to neurons beyond those observed in Alzheimer's disease or Parkinson's disease models. RNA-seq data showed that co-expression of transgenes mainly altered expression levels of genes in the TGF-beta signaling pathway, Wnt signaling pathway, ubiquitin mediated proteolysis, and protein processing pathways in endoplasmic reticulum. Conclusion: Our model might be a useful tool to discover and investigate the etiology of Dementia with Lewy bodies. Moreover, it might be an excellent model to screen drugs that can ameliorate the symptoms of dementia. Acknowledgment: This research was supported in part by the Research grant of Garry Wong MYRG2017-00123-FHS.

Wong, Garry et al. (2019) International Worm Meeting "miRToolsGallery: a microRNA bioinformatics resources database portal."

Wang, Changliang, Knott, K. Emily, Heikkinen, Liisa, Yang, Yang, Chen, Liang, Wong, Garry

[International Worm Meeting, 2019]

microRNAs (miRNAs) are a class of small noncoding RNA derived from primary miRNA transcripts and processed into precursors and eventually to mature miRNAs 21-22 nucleotides in length. miRNAs were originally discovered in C. elegans and are now known to be expressed in animals, plants, and viruses. miRNAs exert their actions as important regulators of cell proliferation, growth, and development via RNA silencing mechanisms. Bioinformatics resources for miRNA study have been developed, but identifying a correct tool for a specific application is tedious and laborious. In order to facilitate this process, we developed a database portal named miRToolsGallery by manual curation of approximately 1000 tools and resources. With miRToolsGallery, a user can easily locate the appropriate tool for an investigation by implementing a searchable and rankable query function. Tools are ranked by different criteria from PageRank algorithm to most recent publication date to number of publications. The portal contains tools covering a broad range of organisms including C. elegans. The tools also encompass an expansive range of applications from target identification to orthology search. Moreover, the tool is a rapid way to locate recent literature within the miRNA bioinformatics field and to identify current trends. One such trend identified with the tool was the stability and frequent use of some very old computational tools, despite the rapid and enormous advancement in knowledge of miRNA function. The resource is available at www.mirtoolsgallery.org. This research was supported in part by the Research grant of Garry Wong MYRG2016-00101-FHS.

Holman, Elizabeth et al. (2021) International Worm Meeting "Autonomous Adaptive Data Acquisition for Scanning Hyperspectral Imaging in Caenorhabditis elegans"

Holman, Hoi-Ying, DeWeese, Michael, Holman, Elizabeth, Sternberg, Paul, Fang, Yuan-Sheng, Chen, Liang

[International Worm Meeting, 2021]

Non-invasive and label-free spectral microscopy (spectromicroscopy) techniques can provide quantitative biochemical information complementary to genomic sequencing, transcriptomic profiling, and proteomic analyses. However, spectromicroscopy techniques generate high-dimensional data; acquisition of a single spectral image can range from tens of minutes to hours, depending on the desired spatial resolution and the image size. This substantially limits the timescales of observable transient biological processes. To address this challenge and move spectromicroscopy towards efficient real-time spatiochemical imaging, we developed a grid-less autonomous adaptive sampling method. Our method substantially decreases image acquisition time while increasing sampling density in regions of steeper physico-chemical gradients. When implemented with scanning Fourier Transform infrared spectromicroscopy experiments, this grid-less adaptive sampling approach outperformed standard uniform grid sampling in a two-component chemical model system and in a complex biological sample, Caenorhabditis elegans. We quantitatively and qualitatively assess the efficiency of data acquisition using performance metrics and multivariate infrared spectral analysis, respectively.

Edlira Yzeiraj et al. (2007) International Worm Meeting "Regulation and expression of collagens and fat metabolism genes in the dbl-1 pathway."

Dr. Cathy Savage-Dunn, Edlira Yzeiraj, Mariya Fabisevich

[International Worm Meeting, 2007]

In C. elegans DBL-1 is a member of the TGF-<font face=symbol>b</font> superfamily closely related to Drosophila Dpp and vertebrate BMP-2/BMP-4 (Suzuki et al., 1999, Development 126, 241-250). The DBL-1 signaling pathway regulates body size and patterning of male-specific copulatory structures. In regulating body size DBL-1 acts as a dosage-dependent regulator (Suzuki et al., 1999). Loss of dbl-1 activity results in smaller animals, while overexpression of dbl-1 results in longer animals. From microarray analysis 242 genes regulated by dbl-1 were identified. From these, 159 genes are repressed but only 83 genes are activated (Liang et al., 2007, Developmental Biology, In Press). We are interested in determining the biological function of some of these putative target genes. We are focusing on collagens and genes involved in fat metabolism. Collagen genes are involved in body size regulation. Cuticle collagens are synthesized by the hypodermis and secreted on the surface of the worm body as an exoskeleton. In the C. elegans genome, about 175 genes encode collagen-like polypeptides. Mutation of these genes causes defects in body morphogenesis, e.g. dumpy, roller, blister, and embryonic lethality (Myllyharju and Kivirikko, 2004, Trends Genet. 20, 33-43). From the microarray analyses three collagen genes were identified as dbl-1-regulated target gene, col-141, rol-6 and col-41. It is believed that dbl-1 represses col-141 and activates rol-6 and col-41 (Liang et. al., 2007). We plan to confirm the gene regulation of these genes by initially performing RT-PCR and then do RNAi to test their function. We are also focusing on genes involved in fat metabolism and transport, specifically vit-5, vit-6, fat-6, fat-7, and ins-7 (Liang et. al., 2007). We are interested in whether the dbl-1 pathway regulates body size in part by regulation of fat storage and metabolism. In addition these genes are believed to play a key role in C. elegans aging. vit-5, vit-6, and ins-7 are upregulated by the dbl-1 pathway, and fat-6 and fat-7 are downregulated by it, and this regulation has been confirmed by RT-PCR for some of these genes. To examine these genes we will use fat staining, GFP-reporters, and RNAi feeding. During the fat staining procedure, worms ingest fluorescent particles, which are temporarily stored in the intestinal cells. The cells can then be seen under a fluorescent microscope, and analyzed for number and degree of fat content. GFP reporters will be used on fat-6, fat-7 and ins-7 to locate the physical expression of the gene.

Frederick J Tan et al. (2004) West Coast Worm Meeting "Functions of the Outer Mitochondrial Membrane Protein CED-9"

R Blake Hill, Andrew Z Fire, Frederick J Tan

[West Coast Worm Meeting, 2004]

CED-9 (CEll Death abnormal) negatively regulates the programmed cell death pathway in C. elegans . This membrane associated pro-survival protein prevents the activation of the CED-3 caspase. Loss of function ced-9 animals arrest at early embryogenesis, presumably dying from ectopic cell deaths. Conversely, gain of function animals appear healthy, albeit with extra cells (Hengartner, Ellis and Horvitz, 1992). These effects have been ascribed to the ability of CED-9 to sequester the CED-4/CED-3 caspase complex at the mitochondria (Chen, et al. , 2000). We hope to understand the structural features of CED-9 that participate in its function. To this end, we are assaying the activity of various CED-9 mutants and derivatives for ability to prevent cell death and respond to appropriate modulating signals. A question that we consider to be central is whether the C-terminal transmembrane domain of CED-9 serves only as a targeting sequence to the mitochondrion, or plays a more active role. We have demonstrated that this domain is essential for mitochondrial localization and plays some role in the function of CED-9. Distinction between localization and other structural and functional roles is in progress. Hengartner MO, Ellis RE and HR Horvitz. 1992. Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356 , 494-499. Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y, and HR Horvitz. 2000. Translocation of C. elegans CED-4 to Nuclear Membranes During Programmed Cell Death. Science 287 , 1485-1489.

S Chen et al. (1999) International C. elegans Meeting "Are Complex N-glycans Essential for the Development of the Nematode C. elegans?"

AM Spence, S Zhou, S Chen, M Sarkar, H Schachter

[International C. elegans Meeting, 1999]

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of hybrid and complex N-glycans. Disruption of the GnT I gene in the mouse results in arrest of embryogenesis. Defects in the synthesis of complex N-glycans have been shown to be associated with several human congenital diseases. We are investigating the role of complex N-glycans in C. elegans . Three C. elegans genes homologous to mammalian GnT I have been identified by searching the genome databases. They are designated gly-12, gly-13 and gly-14 . cDNAs encoding these predicted genes have been cloned and their expression patterns determined. Both gly-12 and gly-14 encode active enzymes while gly-13 does not, towards the substrate tested [Chen et al ., 1999]. To better understand its biological function, we wanted to isolate a GnT I null mutant. We screened worms that were UV irradiated in the presence of trimethylpsoralen(TMP) and isolated a mutant with a 1.6kb deletion of gly-12 . The deletion covers from intron 6 to exon 12, a DNA fragment encoding most of the GnT I C-terminal catalytic domain. The mutants have no obvious phenotypic defects. A Tc1 insertion has been detected within intron 9 of gly-14 . Screening for Tc1 derived deletions is under way. We will create double mutants that lack both gly-12 and gly-14 to test the redundancy of these two GnT I genes. Chen, S., Zhou, S., Sarkar, M., Spence, A. M., and Schachter, H. (1999) J. Biol. Chem. 274 , 288-297.

Ni, J. et al. (2017) International Worm Meeting "The triggering mechanism of C. elegans germline nuclear RNAi at the native targets."

Ni, J., Gu, S., Kalinava, N.

[International Worm Meeting, 2017]

We wish to understand the molecular mechanisms that distinguish "self" and "non-self" DNA in the germline genome of C. elegans. Nuclear RNAi refers to a set of small RNA-guided chromatin-based gene-silencing pathways (e.g., heterochromatin formation and transcriptional silencing), and plays an essential role in genome surveillance in yeast, plants, and animals. We previously identified a large set of genomic loci that are targeted by germline nuclear RNAi in C. elegans [1,2]. The triggering mechanisms at these native targets appear to be highly complex and are poorly understood. We are using two different approaches to resolve this gap. (1) We are identifying aberrant features associated with germline nuclear RNAi-targeted transcripts. To this end, we are using an unbiased RNA-seq approach to characterize both polyA and non-polyA transcripts in both wild type and mutant animals. In addition, subcellular localization of the native target transcripts are being examined using single-molecule FISH and RNA-seq combined with biochemical fractionation. (2) We are using CRISPR-mediated genome editing to test whether the silencing responses at native targets are indeed triggered by the aberrant features that are identified in (1). To date, we have found that "self" and "non-self" transcripts differ in RNA-pocessing, as well as subcellular localization. By using CRISPR, we have identified cis-regulatory elements that are required for the silencing response. Reference: 1. Ni JZ, Chen E, Gu SG. BMC Genomics. 2014. PMID: 25534009 2. Ni JZ, Kalinava N, Chen E, Huang A, Trinh T, Gu SG. Epigenetics Chromatin. 2016. PMID: 26779286

Alicia Melendez et al. (2001) International C. elegans Meeting "Analysis of the C. elegans homolog of the mammalian autophagy gene beclin 1"

Alicia Melendez, David H Hall, Beth Levine

[International C. elegans Meeting, 2001]

Autophagy is the bulk degradation of cytoplasmic components through an autophagosomic-lysosomal pathway. Under stress or nutrient starvation, mammalian cells as well as yeast cells undergo major degradation of proteins and remodelling of cellular compartments. Autophagy is essential for differentiation, survival during nutrient deprivation, and normal growth control; however, nothing is known about autophagy in the development of multicellular organisms. We are analyzing the role of evolutionarily conserved autophagy genes in C. elegans development, by studying the function of the worm beclin1 homolog. The human beclin 1 gene is the first identified mammalian gene to mediate autophagy and also has tumour suppresor and antiviral function (Liang et al., 1999). In yeast, the beclin1 gene was isolated as an autophagy defective mutation, Apg6 , or as a vacuolar protein sorting mutation, Vps11 . Mammalian Beclin 1 promotes autophagy, but not vacuolar protein sorting in autophagy-defective yeast with a targeted disruption of the yeast ortholog, APG6/VPS30 (Liang et al., 1999). Human Beclin1 shares 28% amino acid with the C. elegans Beclin1 (T19E7.3; C. elegans genome sequencing project). We have inactivated the C. elegans beclin1 homologue by injecting T19E7.3 dsRNA or by soaking N2 worms with T19E7.3 dsRNA. beclin1 RNAi-soaked N2 worms arrested at the L1 stage, whereas, the progeny of the injected beclin1 RNAi N2worms reached adulthood with varying defects in the intestine, including the accumulation of large vacuolar structures in the intestine. Ce beclin1 RNAi also decreases the endocytosis of a YP170(yolk protein) ::GFP fusion by oocytes, an assay that has been used to isolate mutants in receptor mediated endocytosis (Grant and Hirsh, 1999). As autophagy is required for survival under stress or nutrient starvation conditions, we hypothesized that autophagy might be required for dauer formation or survival of the dauer larva. To investigate this hypothesis, we injected daf-2 mutants with Ce beclin1 dsRNA. daf-2 mutants are dauer constitutive when raised at the restricted temperature and have an increased lifespan at the permissive temperature (Riddle, 1997). Ce beclin 1 (RNAi); daf-2 double mutants arrested at different stages of dauer formation when grown at the restrictive temperature. Survival of the daf-2 dauers was also affected after beclin1 RNAi. We are now using electron microscopy to determine if autophagy is increased upon dauer formation, and if autophagy is affected in the Ce beclin 1 RNAi mutants. In summary, Ce beclin 1 RNAi-soaked N2 worms arrest at the L1 stage and display intestinal defects, and the Ce beclin 1 RNAi-injected daf-2 worms arrest at different stages of dauer formation. While the molecular basis of these phenotypes is not yet known, these observations suggest a possible role for beclin 1 -mediated autophagy in C. elegans development and in the survival or formation of the C. elegans dauer stage larvae. B. Grant and D. Hirsh, 1999. Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell 1999 Dec;10(12):4311-26. X. H. Liang et al., 1999. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999 Dec 9;402(6762):672-6. D. L. Riddle, in Caenorhabditis elegans II, D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997), pp. 739-768.

Karla Opperman et al. (2007) International Worm Meeting "A structure-function analysis of the C. elegans L1CAMs."

Lihsia Chen, Xuelin Wang, Karla Opperman, Shan Zhou

[International Worm Meeting, 2007]

Cell adhesion molecules play important roles during development and in maintaining tissue integrity. L1CAMs are a family of immunoglobulin (Ig)-like cell adhesion molecules that are important in vertebrate nervous system development. L1CAMs are conserved in C. elegans, and are encoded by the lad-1/sax-7 and lad-2 genes. SAX-7 and LAD-2 have distinct functions in the nervous system despite overlapping neuronal expression. SAX-7 is required to maintain neuronal positioning while LAD-2 participates in axon pathfinding. We are performing a structure-function analysis to determine how both proteins mediate their functions. Because the cytoplasmic tails between both proteins are distinct, we are focusing our analysis on how the cytoplasmic tail regulates L1CAM functions. Indeed, while the extracellular domains of SAX-7 and LAD-2 are conserved, the LAD-2 cytoplasmic tail is completely divergent and does not contain any of motifs conserved in SAX-7 and vertebrate L1CAMs. These motifs include the ankyrin-binding motif, which links L1CAMs to the spectrin-actin cytoskeleton, the PDZ-binding motif, and conserved tyrosine residues that are phosphorylated. Our preliminary results reveal that both conserved motifs, as well as phosphorylation one of the tyrosine residues, which regulates ankyrin binding, all contribute to the function of SAX-7 in neuronal position maintenance. (1) Chen L, Ong B, Bennett V. J Cell Biol. 2001 Aug 20;154(4):841-55. (2) Wang X, Kweon J, Larson S, Chen L. Dev Biol. 2005 Aug 15;284(2):273-91. (3) Sasakura H, Inada H, Kuhara A, Fusaoka E, Takemoto D, Takeuchi K, Mori I. EMBO J. 2005 Apr 6;24(7):1477-88. Epub 2005 Mar 17.