Alan, Jamie K. et al. (2011) International Worm Meeting "The atypical Cdc-42-like GTPases CHW-1 and CRP-1 redundantly regulate axon pathfinding and cell migration."

Lundquist, Erik A., Alan, Jamie K.

[International Worm Meeting, 2011]

Rho family small GTPases are Ras-related small GTPases that regulate cytoskeletal organization, motility, trafficking, and cell migration. Numerous studies have shown that Rho GTPase signaling pathways contribute to proper neuronal axon pathfinding and migration in C. elegans. There are 7 Rho GTPases in C. elegans: RHO-1 (RhoA), CDC-42 (Cdc-42), CED-10 (Rac1), RAC-2 (Rac1), MIG-2 (RhoG), and the atypical Cdc-42-like molecules CHW-1 (Chp/Wrch-1), and CRP-1 (putative TC10, TCL). Despite extensive knowledge of Rho/Cdc-42/Rac GTPases, little is known about the contribution of the C. elegans atypical Cdc-42-like family members CHW-1 and CRP-1 to axon pathfinding and neuronal migration. CHW-1 is similar to both Chp (Wrch-2, RhoV) and Wrch-1 (RhoU), while CRP-1 is similar to both TC10 (RhoQ) and TCL (RhoJ). CHW-1 and CRP-1 are predicted to, or have been shown to display altered GDP/GTP cycling characteristics (Jenna et al., 2005). CHW-1 contains an alanine instead of the conserved glycine at the 12 position (using Ras numbering), which is predicted to be a partially activating mutation. CRP-1 resembles TC10 and TCL, and like these GTPases, CRP-1 also shows altered GDP/GTP binding characteristics (Neudauer et al., 1998; Vignal et al., 2000; Jenna et al., 2005). To test the roles of CHW-1 and CRP-1 in axon pathfinding and cell migration, we assayed the PDE axons and the migrations of the Q descendants AQR and PQR in chw-1(ok697) and crp-1(ok685) mutants. We found that loss of chw-1 or crp-1 alone resulted in only modest pathfinding defects in PDE neurons, similar to cdc-42(gk388). However loss of both cdc-42 and chw-1 or chw-1 and crp-1 resulted in synergistic increases in PDE axon pathfinding defects. These results suggest that, similar to the Rac-like GTPases CED-10 and MIG-2 (Lundquist et al., 2001), the Cdc-42-family GTPases CDC-42, CHW-1, and CRP-1 act redundantly in axon pathfinding. Preliminary studies indicate that loss of either chw-1 or crp-1 result in modest defects in AQR migration, suggesting that they might redundantly control cell migration as well. As a complementary approach, we plan to drive the expression of mutant versions of CHW-1 and CRP-1 in the PDE neurons and AQR/PQR to determine the effects of overactivity, similar to studies on the Rac GTPases (Struckhoff and Lundquist, 2003). Preliminary results indicate that expression of a mutant CHW-1 transgene predicted to have increased GTPase activity disrupted neuronal morphology. Taken together, these data provide preliminary evidence for a role of the atypical Cdc-42-like GTPases CHW-1 and CRP-1 in axon pathfinding and neuronal migration.

Alan, Jamie K. et al. (2013) International Worm Meeting "The atypical Rho GTPase CHW-1 works downstream of SAX-3/Robo to mediate axon guidance in Caenorhabditis elegans."

Alan, Jamie K., Lundquist, Erik L.

[International Worm Meeting, 2013]

During development, neuronal cells extend an axon towards their target destination in response to a cue to form a properly functioning nervous system. Rho proteins are Ras-related small GTPases that regulate cytoskeletal organization and dynamics along with cell adhesion and motility. Furthermore, Rho proteins such as CDC-42, MIG-2 and CED-10 are known to regulate axon guidance. Despite extensive knowledge about canonical Rho proteins (RhoA/Rac1/Cdc-42), little is known about the C. elegans atypical Cdc-42-like family members CHW-1 and CRP-1 in regards to axon pathfinding and neuronal migration. chw-1(Chp/Wrch) encodes for a protein that resembles human Chp (Wrch-2/RhoV) and Wrch-1 (RhoU), and crp-1 encodes for a protein that resembles TC10 and TCL. Here, we show that chw-1 works redundantly with crp-1 and cdc-42 in axon guidance. Furthermore, our experiments suggest that precise levels of chw-1 expression and activity are required for proper axon guidance. Also, chw-1 genetically interacts with the guidance receptor sax-3. Finally, in VD/DD motor neurons chw-1 and sax-3 might act together to control axon guidance. In summary, this is the first studying implicating the atypical Rho GTPases chw-1 and crp-1 in axon guidance. Furthermore, this is the first evidence of a genetic interaction between chw-1 and the guidance receptor sax-3. Finally, we show that chw-1, but not crp-1, might act with sax-3 in axon guidance.

Sucharski, Holly et al. (2015) International Worm Meeting "Exploring the role of axon guidance molecules in pancreatic cancer using C. elegans."

Nichols, Rodney, Kagande, Axucillia, Gardner, Morgan, Sucharski, Holly, Alan, Jamie, Coombs, Seth

[International Worm Meeting, 2015]

Pancreatic cancer is highly lethal, with a <5% 5-year survival rate. Therefore, there is a critical need to identify effective therapies to treat pancreatic cancer. Recently, a study identified aberrations in axon guidance genes, including SLIT/ROBO signaling, in pancreatic cancer. There is evidence to suggest that SLIT/ROBO signaling is both upregulated and downregulated in cancer. However, very little is known about how these molecules contribute to carcinogenesis and metastasis. Therefore, there is a critical need to uncover the roles of axon guidance molecules in tumorigenesis and metastasis. Specifically, we aim to uncover the signaling pathways involved in aberrant SLIT/ROBO signaling in pancreatic cancer. To do this we will turn to the model organism C. elegans. Signaling pathways in C. elegans are well conserved, and C. elegans have been previously used to elucidate important molecules in oncogenic signaling pathways. The orthologue of ROBO in C. elegans is sax-3, which encodes a transmembrane protein involved in axon guidance. To explore the pathways that are important when sax-3 is upreguated, a gain-of-function transgenic strain expressing activated sax-3 (myr-sax-3) in the VD/DD motor neurons will be used. This mutation causes profound axon pathfinding defects. Using RNAi, we knocked down candidate genes in the myr-sax-3 strain. If these genes are required downstream of activated sax-3, then the pathfinding defects will be attenuated. To explore the pathways that are important when sax-3 is downregulated, a deletion strand of sax-3, sax-3(ky123), was used in a synthetic lethal (syn/let) screen to determine which genes are required in the absence of sax-3. The syn/let screen uses a loss-of-function but viable sax-3 deletion strain (sax-3(ky123)) to test if the loss of any genes involved in a parallel or downstream pathway will result in a synthetic/lethal phenotype. Genes were identified for this screen based on the C. elegans orthologues of human genes that are associated with pancreatic cancer and cancer cell phenotypes such as migration and invasion. Results from the gain-of-function experiment yielded some candidate genes such as exc-5 and chw-1. Results from the loss-of-function experiment yielded some candidate genes including let-60/ras, ptp-3/lar (leukocyte common antigen related), and gsk-3 (glycogen synthase kinase 3 beta). It is important to note that oncogenic activating mutations in ras have been found in 90% of pancreatic cancer patients. In the future, the genes identified in this screen will be verified using human pancreatic cancer cells. .

Nariya, Hardik et al. (2017) International Worm Meeting "An investigation of the effects of artificial light at night on C. elegans offspring production and lifespan."

Buchanan, Bryant, Wise, Sharon, Zhushma, Rosa, Nariya, Hardik, Shinn-Thomas, Jessica

[International Worm Meeting, 2017]

Artificial light at night (ALAN) has many broad-scale and global implications for ecosystems and wildlife that have evolved under a 24-h circadian cycle. With increased urbanization, artificial light at night has directly altered natural photoperiods and nocturnal light intensity. Artificial light at night can disrupt behavioral patterns such as foraging activity and mating in animals. Disturbances in natural light and dark cycles also affect melatonin-regulated circadian and seasonal rhythms in Drosophila. We investigated the impact of ecologically relevant levels of light pollution on an important invertebrate model, Caenorhabditis elegans, as the impact of night lighting at these light levels is currently unknown. In this study, we exposed worms to artificial light at four intensities: 10-4 lx (control, comparable to natural nocturnal darkness), 10-2 lx (comparable to full-moon lighting and a low level of light pollution), 1 lx (comparable to dawn/dusk or intense light pollution), and 100 lx (dim daylight level comparable to extreme light pollution) on a 12L:12D photoperiod (100 lx treatments experienced constant light). We measured the impact of these light treatments on offspring production in hermaphroditic C. elegans. We grew worms for 2 generations in each light treatment, and then recorded the lifespan and counted the number of hatched offspring produced in the F3 generation. Our data show no significant differences among light levels for lifespan or offspring production suggesting that at least for these life history traits, ALAN does not affect these soil nematodes. Future directions include measuring additional life history traits and circadian gene expression for worms exposed to ALAN.

sarparast, Morteza et al. (2021) International Worm Meeting "Soluble Epoxide Hydrolase Inhibitor, AUDA, Recuses Neurodegeneration Induced by Amyloid beta and Tau"

Sarparast, Morteza, Zhang, Fan, Ramirez, Leslie, Stephen Lee, Kin Sing, K. Alan, Jamie, Kessler, Benjamin

[International Worm Meeting, 2021]

According to a United Nations report, the percentage of the population over the age of 65 is expected to increase from approximately 9% (2019), to roughly 20% by 2050. With this demographic change, we can expect a coinciding increased incidence of age-related neurodegenerative diseases (ND), including Alzheimer's Disease (AD), for which there is no cure. The two hallmark pathologies of AD are the deposition of the amyloid beta (Abeta) and the neurofibrillary tangles of the microtubule-binding protein tau. Recent evidence suggests that increasing the epoxy-metabolites of polyunsaturated fatty acids (PUFAs) through pharmacological inhibition or genetic knock-out of soluble epoxide hydrolase (sEH), which metabolizes epoxy-PUFAs, could be an effective strategy in limiting AD neurodegeneration. Considering this, we seek to investigate the effect(s) of epoxy-PUFAs on tau- and Abeta-induced neurodegeneration using Caenorhabditis elegans (C. elegans). The transgenic Tau (CK1441) and Abeta (CL2355) mutants show neurodegenerative behavior such as slow thrashing and decreased locomotion speed, respectively, and both strains show hypersensitivity to serotonin. Interestingly, supplementation of HE inhibitor, 12-[[(tricyclo[3.3.1.13.7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid (AUDA), rescues neurodegeneration in both transgenic strains. Our results suggested that specific epoxy-PUFAs significantly affect neurodegeneration, in particular those mediated by deposition of Abeta and/or tau, and the mechanistic studies on how EH inhibition alleviates neurodegeneration induced by Abeta and/ or tau are underway.

Kessler, Benjamin et al. (2021) International Worm Meeting "Disrupting Polyunsaturated Fatty Acid Biosynthesis Modulates Lifespan and Healthspan"

Sarparast, Morteza, Lee, Kin Sing Stephen, Dattmore, Devon, Alan, Jamie K., Kessler, Benjamin, Zhang, Fan

[International Worm Meeting, 2021]

Specific omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) have protective effects against aging-related conditions such as cardiovascular disease, inflammation, and neurodegenerative diseases. However, it is unclear which PUFAs are required in the diet and how they affect human health and disease. This study will investigate the physiological roles of individual PUFAs in order to pharmacologically and dietarily promote healthy aging. We will use fatty acid desaturase enzyme knockout transgenic C. elegans strains to assess the in vivo effects of PUFAs on the aging process. This investigation will create a dataset that includes lifespan and healthspan data (as determined by thrashing and/or egg laying) for every available fatty acid desaturase enzyme genetic knockout in the worm. Additionally, lipidomic or metabolic analysis will be used to assess the lipidome of key strains. We hypothesize that specific PUFAs modulate physiological processes through their corresponding downstream metabolites' role in lipid signaling. Our data showed that mutants with genetically disrupted PUFA biosynthesis displayed a decreased median lifespan, poor physical fitness, and altered egg laying patterns. Interestingly, our results from several mutants are different from published data involving the use of FuDR, a chemical that prevents progeny. It is expected that lipidomic analysis of the knockout worms will reveal that our in vivo observations are associated with an altered lipid panel, especially downstream metabolites. Our data suggest that genetically altering endogenous levels of PUFAs modulates lifespan and healthspan. However, specifically limiting omega-3 PUFA biosynthesis has a rescuing effect on healthspan. Investigating these metabolic pathways may elucidate novel drug targets that could revolutionize chronic disease treatment and prevention.

Lee, Kin Sing Stephen et al. (2021) International Worm Meeting "The Effect of Cytochrome P450 Metabolites of Dietary Polyunsaturated Fatty Acids on Age-Associated Neurodegeneration."

Kessler, Benjamin, Pourmand, Elham, Lee, Kin Sing Stephen, Sarparast, Morteza, Alan, Jamie, Ramirez, Leslie, Zhang, Fan

[International Worm Meeting, 2021]

By 2035, the population older than 65 is projected to be 21.3% (78 million) increasing from 15.2% in 2016. Most elderly patients eventually develop some degree of cognitive dysfunction, and Alzheimer's disease alone affects almost 33% to 50% of the population over 85. As such, there is an unmet medical need to identify pathways that could curb neurodegeneration. Such a discovery significantly improves the quality of life for elderly patients. Recent research demonstrated that inhibition of cytochrome P450 (CYP) metabolism of dietary polyunsaturated fatty acids (PUFAs) is beneficial for several neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. However, the detailed mechanism by which specific PUFA CYP metabolites responsible for such beneficial effects remains largely unknown. To tackle this problem, we apply a multidisciplinary approach to investigate the underlying mechanism and identify the signaling molecules responsible for the effects of PUFA CYP metabolites on neurodegeneration. We will use C. elegans as an animal model because it has been used to investigate neurodegeneration mechanism for more than a decade. Specifically, in this presentation, we will present our efforts in developing and applying different chemical tools to study the mechanism of how PUFA CYP metabolites affect neurodegeneration in different transgenic C. elegans strains. We found that inhibition of epoxide hydrolase in C. elegans rescues the neurodegenerative phenotypes in the strains expressing either amyloid-beta or tau, which are similar to what has been found in the murine Alzheimer's disease amyloidosis or tauopathy models. We also demonstrated that a specific omega-6 PUFA CYP metabolite actually induces neurodegeneration. This result is interesting as most studies suggested that in general, PUFA CYP metabolites are beneficial for neurodegenerative disease. Our studies open new directions for neurodegeneration and C. elegans research, and further mechanistic studies are currently being undertaken in our laboratory.

Shownkeen R et al. (1995) International C. elegans Meeting "THE CAENORHABDITIS ELEGANS PHYSICAL MAP"

Chissoe SL, Waterston RH, Fraser A, Sulston JE, Shownkeen R, Coulson AR

[International C. elegans Meeting, 1995]

Twelve contigs of cosmids and yeast artificial chromosomes (YACs) span more than 95Mb of the 100Mb C.elegans genome. 650 markers link the physical and genetic maps.Hybridisation of tag-sequenced cDNA clones to a map-representative set of YACs indicates that the map incorporates in excess of 99.8% of genes. The map is accessible in ACeDB. We (S.C.) are investigating the representation by bacterial artificial chromosomes (BACs) of regions of the genome not represented by cosmids. Two grids of YACs, of 958 clones ('Poly2') and 223 clones ('Suppoly') are available on request. The latter represents regions of the genome that have been characterised or better defined since the selection of clones for the former. Cosmid clones and YAC grids are available from the Sanger Centre (requests to alan@sanger.ac.uk; FAX 01223 494919). YAC clones and 'cm' series cDNA clones are available from the Sanger Centre or Washington University (rw@nematode. wustl.edu; FAX 314 362 2985).

Chan S et al. (1991) International C. elegans Meeting "SEEKING UNC-40."

Culotti JG, Su M-W, Chan S, Hedgecock EM

[International C. elegans Meeting, 1991]

unc-40 is required with unc-6 to guide ventral migrations on the nematode epidermis (Hedgecock et al., Neuron 4, 61-85, 1990). We have positioned unc-40 genetically by 3-factor crosses with dpy-5 and bli-4 as flanking markers and we have isolated 5 new alleles of unc-40 (2 spontaneous, 2 gamma, and 1 EMS) making a total of 12. DNAs from cosmids covering this region (obtained from Alan Coulson and John Sulston) are being used to probe DNA and RNA from the mutants and DNA from strains carrying duplications that break to either side of unc-40 (obtained from Anne Rose's laboratory). We are also injecting cosmid DNAs into gonads to look for rescue by germline transformation. The brood sizes of various unc-40 alleles are very small, so we have resorted to injecting the wild type in order to create a series of transformed lines, each carrying an extrachromosomal array of a different cosmid. Each array will be passed into appropriately marked unc-40 animals to ask if any can rescue the mutant phenotype. Mosaic experiments are also underway to determine whether unc-40 acts in migrating cells.

Beitel GJ et al. (1991) International C. elegans Meeting "CLONING AND SEQUENCING OF THE SYNTHETIC MULTIVULVAL GENE lin-9"

Beitel GJ, Horvitz HR

[International C. elegans Meeting, 1991]

A synthetic Multivulva phenotype can result from mutations in two separate genes, while each mutation alone results in an apparently wild-type phenotype (Ferguson and Horvitz, Genetics 123:109-121,1989). Mutations that can cause this synthetic Multivulva (syn Muv) phenotype have been grouped into two classes, A and B, such that a double mutant carrying one mutation in each class will have the syn Muv phenotype. Iin-9(nll2) III is a class B mutation that results in an apparently wild-type phenotype by itself. We previously reported that ZK637 and overlapping cosmids can rescue lin-9 (WBG 11(2 ): p 29,1990). We have now identified an 8 kb subclone with rescuing activity. Using this 8 kb fragment to probe Stuart Kim's cDNA library, six positive plaques representing at least three independent clones were isolated. This 8 kb probe also detects a message of approximately 2.5 kb on Northern blots. The message level appears similar in eggs and mixed-stage populations. We are currently sequencing these cDNAs and looking for evidence that these transcripts do in fact represent the lin-9 gene. This work will be greatly aided by the genomic sequence provided by Molly Craxton, John Sulston and Alan Coulson, as ZK637 was fortuitously chosen as one of the first cosmids to be sequenced in the worm genome sequencing project.