Doyle, James-Julian et al. (2015) International Worm Meeting "C. elegans model of PGRN-deficient FTLD."

Parker, Alex, Bennett, Hugh, Doyle, James-Julian, Bateman, Andrew

[International Worm Meeting, 2015]

Frontotemporal lobar degeneration (FTLD) is a devastating neurodegenerative disease, characterized by the progressive atrophy of the frontal and temporal lobes of the brain. Advances in genetic testing have identified many of its underlying genetic causes. Among the genes identified are mutations in the GRN gene encoding for Progranulin (PGRN). To date over 70 different mutations have been identified, most resulting in a haploinsufficiency of the protein. Recent studies have shown that PGRN has neuroprotective properties, protecting against mutant TDP-43 and polyglutamine toxicity, and plays a significant role in cell survival and stress resistance. Nonetheless, its normal function in the brain, as well as its pathogenic mechanism, remain poorly understood. In order to understand the mechanism of PGRN-deficient FTLD, our lab has turned to the C. elegans nematode as a model. Given that C. elegans have an orthologue of the human GRN gene, pgrn-1, we used the pgrn-1(tm985) deletion mutant to model the loss-of-function. We began by characterizing the C. elegans pgrn-1(tm985) phenotype. This revealed that the worms present a motility defect when assessed in liquid culture. In order to better understand the action of PGRN-1 in the worms, we used RNAi knockdown technology to supress gene expression in the worms' muscle, intestinal, and neuronal cells. Furthermore, we have expressed GFP in the mutant worms' GABAergic motor neurons to assess neuronal health. Finally, we have taken advantage of the worm as a genetic tool to study the interactions between pgrn-1 and the worm orthologues of other known FTLD-causative genes. Based on our findings thus far, we will utilize the worm motility defect to our advantage by performing a high-throughput drug screen to identify potential therapeutics. Together, our findings will increase current understandings on the cause of PGRN-deficient FTLD.

Doyle, James Julian et al. (2017) International Worm Meeting "C. elegans chemical genetic screen to counter Progranulin deficiency."

Parker, Alex, Maios, Claudia, Vrancx, Celine, Doyle, James Julian, Bateman, Andrew, Bennett, Hugh

[International Worm Meeting, 2017]

Progranulin (PGRN) is a widely-studied neurotrophic factor known for its ability to reverse toxicity induced by amyloid beta , TDP-43, and Huntingtin, and is an attractive therapeutic target for its role as a neuronal survival factor. Mutations in the encoding gene result in either frontotemporal dementia or neuronal ceroid lipofuscinosis. Despite the important role of this protein in the maintenance of neuronal health, there are no approved therapeutics capable of modulating the downstream molecular action of PGRN. Since performing large-scale chemical screens in vivo are costly and unfeasible in rodents, screening in C. elegans is an attractive approach to address this issue. Characterization of pgrn-1 mutant worms indicated they displayed an age-dependent paralysis, an effect which can be accelerated when they are placed in liquid culture allowing for large scale, rapid screening. We screened ~4000 commercially-available small molecules in liquid culture for their ability to rescue pgrn-1(-)-induced motility defects, and validated them by their ability to also rescue age-dependent paralysis. In total, we have identified 12 compounds as hits capable of specifically restoring pgrn-1 deficiency, and we will further assess the preclinical efficiency of these compounds in zebrafish and cell culture models. This screen represents the first step in a collaborative effort to translate compounds from the bench to the clinics.

KA Kuznicki et al. (1999) International C. elegans Meeting "Unwinding the P granules: evidence from RNAi and glh mutants"

KL Bennett, JW Kirchner, PA Smith, KA Kuznicki

[International C. elegans Meeting, 1999]

The Bennett laboratory studies the four g erm l ine RNA h elicases (GLHs) , components of the C. elegans P granules. The GLHs are members of the DEAD-box family of RNA helicases and contain retroviral-like CCHC zinc fingers and uncharged glycine-rich repeats (Roussell and Bennett 1993 PNAS 90 :9300-9304, Gruidl et al . 1996 PNAS 93 :13837-13842). GLH-3 and GLH-4, more recently discovered helicases, differ from GLH-1 and GLH-2; GLH-3 lacks the uncharged glycine-rich repeats of the others, while GLH-4 is the most unique. RNAi implies that each GLH has a different role. RNAi of glh-1 or glh-2 produces sterility in a small percentage of the F 1 generation at the restrictive temperature. By contrast, glh-3 RNAi causes smaller brood sizes in the injected worm at the restrictive temperature. Using a combination of the helicase regions of glh-1 , glh-2 , and glh-3 results in a more dramatic temperature sensitive phenotype. Approximately 75% of the F 1 generation is infertile. Other combinatorial glh RNAi injections will further determine if the glhs function synergistically. A glh-3 - strain with a 1.7 kb deletion was identified in our laboratory from a TMP/UV library we generated. Preliminary analysis of the glh-3 - strain indicates that it has a mild temperature sensitive fertility defect, with brood size approximately 60-70% of that seen with wild type worms at 25-26degC. This result is consistent with that seen in the glh-3 RNAi experiments. Ongoing experiments include glh-4 RNAi and screening for additional glh mutants.

Kreutzer MA et al. (1995) International C. elegans Meeting "CHARACTERIZATION OF THE CAENORHABDITIS ELEGANS CYCLIN GENES"

Richards JP, Bennett KL, Kreutzer MA

[International C. elegans Meeting, 1995]

The Bennett laboratory has identified genes that may be essential for nematode germline determination. Drosophila cyclin B RNA has been shown to localize to the Drosophila polar granules, germ granules that many be equivalent to the nematode P granules. Therefore, cyclins may play a role in the development of the germline. We have cloned cDNAs for the C. elegans cyclins A1, B and B3 (Kreutzer, et al. in press). Cyclins A1 and B are most closely related to either A- or B- type cyclins of other species while cyclin B3 is a distant ancestral cyclin gene. While cyclin A1 is a member of a large A-type multigene family that corresponds to a single transcript on northern blots, cyclin B3 appears to be a single gene recognizing a single transcript. In contrast, cyclin B recognizes three transcripts with one cyclin B transcript specific for the paternal germline. Our results suggest that the largest and smallest cyclin B transcripts use different polyadenylation sites. By northern analyses, the two other cyclin B as well as the cyclin A1 and cyclin B3 transcripts are most abundant in the maternal germline. In addition, two types of 3'UTR translational regulatory elements, ACEs and NREs, are found in the 3' untranslated region of each of these genes. We are currently using probes to the cyclin genes for in situ hybridization to wild type and germline defective adult worms and embryos to determine the localization of these RNAs during germline proliferation and embryogenesis.

Te-Wen Lo et al. (2007) International Worm Meeting "C. elegans FGF Receptor Signaling Can Occur via Direct Binding of the SEM-5/Grb2 Adaptor Protein."

Michael Stern, Te-Wen Lo

[International Worm Meeting, 2007]

The components of receptor tyrosine kinase(RTK)signaling complexes help to define the specificity of the effects of their activation. The C. elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2 binding motifs present in the CTD (Y1009 and Y1087), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. Although SEM-5 is required for the fluid homeostasis function of EGL-15, these sites are not required for that function, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multisubstrate adaptor protein FRS2 serves to link vertebrate FGFRs to GRB2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation1 but is not required for EGL-15 function2 . Thus, unlike the vertebrate FGFRs, which require the multisubstrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly. 1Matusbara et al., 2007. Genes to Cells; 12(3): 407-420. 2see D. Bennett abstract.

Chen LS et al. (1997) International C. elegans Meeting "MOLECULAR AND GENETIC ANALYSIS OF C. ELEGANS NEF-1, A MEMBER OF THE IG/FNIII SUPERFAMILY OF CELL ADHESION MOLECULES"

Bennett V, Chen LS

[International C. elegans Meeting, 1997]

A cell's identity, its interactions with other cells, and its ablity to adapt to its environment are heavily dependent on the proteins associated with its plasma membrane. Ankyrin, an intracellular protein that couples several integral membrane proteins to the spectrin cytoskeleton, is believed to be instrumental in organizing many membrane proteins into specialized domains. Recently, a family of cell adhesion molecules belonging to the Ig/FnIII superfamily was shown to bind ankyrin; these proteins include neurofascin, L1, NrCam and NgCam in vertebrates and neuroglian in Drosophila (1-6). This family of ankyrin binding CAMs is believed to be involved in neurite outgrowth, axonal fasciculation and targeting, cell migration and synaptogenesis during embryonic and postnatal vertebrate development (1-4). A member of the family has been identified in C. elegans. The gene encoding the homologue has been designated nef-1. In addition to the Ig and FnIII domains, it contains a highly conserved ankyrin binding domain. We are interested in elucidating the role of nef-1 in C. elegans development and its interactions with the C. elegans ankyrin homologue, UNC-44. Interestingly, mutations in unc-44 causes defects in axon guidance (7). 1. Sonderegger and Rathgen, 1992. J.Cell Biol. 119:1387-1394. 2. Rathgen and Jessel, 1991. Sem. of Neurosci. 3:297-307. 3. Grumet, 1991. Curr. Opin. Neurobiol. 1:370-379. 4. Hortsch and Goodman, 1991. Ann. Rev. Cell Biol. 7:505-557. 5. Davis et al., 1993. J. Biol. Chem.232:121-133 6. Davis and Bennett, 1994. J. Biol. Chem.267:18955-18972. 7. Otsuka et al., 1995. J.Cell Biol. 129: 1089-1092.

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.

Smith PA et al. (1996) Midwest Worm Meeting "C. elegans germline RNA helicases: are they all components of the P granules?"

McCrone JS, Smith PA, Bennett KL, Kuznicki KA, Kirchner JW, Strome S, Gruidl ME

[Midwest Worm Meeting, 1996]

The glh genes are a family of putative germline RNA helicases in C. elegans. glh-1 has been characterized as a germline-specific transcript coding for a predicted protein with eight conserved RNA helicase motifs, glycine-rich imperfect repeats in the N terminus, and four retroviral-like CCHC zinc fingers (Roussell and Bennett, 1993, PNAS USA 90:9300). Antibodies against two regions of GLH-1 fusion proteins recognize the germline-specific P granules of C. elegans throughout all stages of worm development. GLH-1 protein is uniformly distributed in the cytoplasm of the oocyte and segregates with the germline precursor cell from the first embryonic cell division. By Western analysis a 78kD worm protein, the predicted size for GLH-1, is seen. The closely-related glh-2 gene is also germline-specific, maps near glh-1 and codes for a less abundant, larger mRNA. The in situ hybridization patterns of glh-1 and glh-2 differ. The presence of glh-1 RNA is associated with regions of the hermaphrodite gonad where germ cell proliferation, oogenesis, and spermatogenesis occur. glh-2 mRNA is largely confined to the region where oogenesis occurs; however, glh-2 RNA is not associated with spermatogenesis. glh-2 mRNA is also detected in young C. elegans embryos. The predicted GLH-2 protein differs from GLH-1 in having six zinc fingers and different glycine repeats. Preliminary studies with GLH-2 antibodies indicate GLH-2 is present in oocytes and in P granules. The Genome Sequencing Center has uncovered an additional region on LG1 that closely matches glh-1 and glh-2. This potential GLH-3 contains two somewhat divergent CCHC fingers, but lacks the glycine repeats. Questions remain. Are all GLH proteins localized to the P granules? Do their roles differ in the germline?

Lasse, Samuel et al. (2015) International Worm Meeting "Loss of tax-4 function alters the temperature-dependence of egg-laying."

Goodman, Miriam, Koulechova, Diana, Lasse, Samuel

[International Worm Meeting, 2015]

The temperature dependence of muscle function has been well documented 1. Whether this dependence is due to the intrinsic properties of muscles, their control by motor neurons, regulatory input from thermoreceptor neurons, or some combination of these factors is unclear. We are investigating this question using the nematode C. elegans and egg-laying behavior. We first measured steady-state egg-laying rate as a function of temperature in several wild strains isolated from a range of climates and used these data to estimate the peak egg-laying rate (Rmax) and the temperature at which it occurs (Tmax). We find that Tmax, but not Rmax correlates with the distance from the equator for each strain's origin. In a companion presentation (Lasse, McPherson, et al), we share initial work directed towards identifying genetic polymorphisms that might account for the variation in Tmax, focusing on the two most divergent strains: Bristol (N2) and Hawaiian (CB4856). To learn more about the contribution of thermoreceptor neurons, we leveraged existing and novel mutations in the tax-4 gene, which is required for the function of all thermoreceptor neurons operating in the innocuous thermal zone2. In both Bristol (N2) and Hawaiian (CB4856) strains, loss of tax-4 function decreases Tmax. This manipulation reduces Rmax by a factor of three in the Bristol (N2) background but does not alter Rmax in the Hawaiian (CB4856) background. We propose that the motor program has similar intrinsic temperature dependence across worm strains and that thermoreceptor neuron input is necessary to shift this dependence to a range that is best matched to a particular environment.We thank Mario de Bono and Changchun Chen for assistance with the CRISPR technique and the NIH/IRACDA for fellowship funding to S. Lasse.1. Bennett, A. F. Thermal dependence of muscle function. Am. J. Physiol. 247, R217-29 (1984).2. Mori, I. & Ohshima, Y. Neural regulation of thermotaxis in Caenorhabditis elegans. Nature 376, 344-348 (1995).

Katherine M Walstrom et al. (2003) International Worm Meeting "RHA-1 is Required for Germ Cell Proliferation"

William G Kelly, Deborah Schmidt, Katherine M Walstrom

[International Worm Meeting, 2003]

RNA helicase A (RHA) is conserved in many organisms and has numerous cellular functions. Human RHA is required for RNA export from the nucleus (Tang et al. (1999) MCB 19, 3540) and the Drosophila homolog, MLE, is required for dosage compensation in male flies (Kuroda et al. (1991) Cell 66, 935). We are characterizing the C. elegans strain tm329 that contains a deletion in rha-1, the C. elegans homolog of RHA. These worms develop normally at 16-17 degrees C, but at 25.5 degrees C, hermaphrodites and males have small gonads containing few germ cells, most with abnormal nuclear morphology. Staining of the mutant gonads with an antibody against a mitosis marker (phosphorylated serine 10 on histone H3) demonstrates that there are fewer cells undergoing mitosis in the mutant worms compared to N2 worms. The germ cells are not degraded by apoptosis (tested using uptake of acridine orange). Meiotic cells were labeled in the mutants with an antibody against HIM-3. Meoisis starts in the middle of the mutant gonads but does not proceed properly, and few gametes are formed. At least 80% of both male and hermaphrodite rha-1(tm329) worms grown at 25.5 degrees C are sterile, with the fertile worms producing approximately 10 offspring, compared to 140 for N2 worms grown under the same conditions. To test whether rha-1 is involved in a known germline pathway, rha-1(tm329) embryos and young adult germlines were stained with antibodies directed against the P-granule proteins GLH-1 and PGL-1. Both proteins were properly distributed throughout the development of the rha-1(tm329) mutants, and the numbers of P-granules in embryos and around the germ cell nuclei in both mutant and wild-type worms were similar. The rha-1(tm329) mutant worms have some phenotypic similarities to Mes mutants, namely a maternal effect phenotype, underproliferated gonads, and similar abnormal germ cell nuclear morphologies. Additional experiments testing for other similarities between Mes mutants and rha-1(tm329) mutants will be discussed. We are grateful to the C. elegans Gene Knockout Consortium for the tm329 strain and to K. Bennett, S. Strome, and M. Zetka for antibodies.