Nicholas J D Gower et al. (2002) European Worm Meeting "Regulation of inositol 1,4,5-trisphosphate receptor gene (itr-1) expression by pha-4"

Nicholas J D Gower, Howard A Baylis

[European Worm Meeting, 2002]

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a central role in the organisation of calcium signals by regulating intracellular calcium flux. IP3Rs in C. elegans are encoded by a single gene, itr-1 (also called lfe-11 and dec-42). We have shown previously that the itr-1 gene has three putative transcriptional start sites3. Sequence comparisons to the C. briggsae itr-1 gene supported the hypothesis that itr-1 has three promoters (pA, pB and pC), each of which gives rise to an alternative mRNA and hence unique protein. By using transgenic GFP reporter constructs we showed that these promoters direct cell-specific expression of itr-1 variants and further that this results in adjacent cells in the same tissue containing different IP3R isoforms4.

AK Jones et al. (2009) European Worm Neurobiology Meeting "A mutation in the Caenorhabditis elegans nicotinic acetylcholine receptor a subunit, unc-63, provides a model for fast channel congenital myasthenia"

D Rayes, AK Jones, G Hernando, R Jones, SD Buckingham, DB Sattelle, A Al-Diwani, C Bouzat, TPR Maynard

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC and OXION. Abstract: The nematode Caenorhabditis elegans is an established model organism for studying neurobiology since many synaptic molecular components are conserved both in the worm and humans. The unc-63 a subunit of the C. elegans nicotinic acetylcholine receptor (nAChR) plays an important role in fast cholinergic synaptic transmission at the nematode neuromuscular junction (1). Here we show that worms with the unc-63(x26) allele, with its .C151Y mutation that disrupts the Cys-loop, have deficient muscle function as displayed by impaired thrashing locomotion compared to wild-type worms (2). Single-channel recordings from muscle cells of the mutant strain show reduced number of active patches, a 100-fold reduced frequency of opening events, and abnormally reduced duration of channel openings similar to that observed in patients with fast-channel congenital myasthenia syndromes (FCCMS). Interestingly, a mutation in the equivalent position of the e subunit of the human muscle nAChR (.C128S) is associated with a FCCMS due to nAChR deficiency at the endplate (3). Therefore, disruption of the Cys-loop results in qualitatively similar actions on both nematode and human receptors. Therapy for FCCMS is limited in variety and success with 3,4-diaminopyridine (3,4-DAP) and anticholinesterase drugs such as pyridostigmine bromide (PB). We show that PB and 3,4-DAP can partially rescue the motility defect seen in unc-63(x26). Conclusions: We describe the first candidate C. elegans model for screening for drugs aimed at ameliorating the consequences of mutations in nAChRs associated with FCCMS. References: (1) Culetto E et al. J Biol Chem. 2004. 279:42476-83. (2) Jones AK et al. Hum Mol Genet. Submitted 2009. (3) Milone M et al. Ann N Y Acad Sci. 1998. 841:184-8.

LeBlanc, M. et al. (2019) International Worm Meeting "Burrowing as a Genetic Screen of Neuromuscular Functioning in Caenorhabditis elegans."

LeBlanc, M., Vanapalli, S., Rahman, M.

[International Worm Meeting, 2019]

Neuromuscular disorders such as Becker muscular dystrophy (BMD), Duchenne muscular dystrophy (DMD) or amyotrophic lateral sclerosis (ALS) affect the motor neuron or muscles, and causes the muscle to become weak and waste away. There can be different causes for these diseases. Many of them are genetic and have no cure. The nematode, C. elegans is a premier genetic model sharing remarkable similarity with the mammalian neuromuscular system. Additionally, it has long been used as a model to understand programmed cell death. C. elegans is heavily used to understand the genetic mechanism and molecular basis of neuromuscular functions such as locomotion, behavior and muscle strength. The nematode's neuromuscular functioning can be assessed through three simple methods, by scoring (i) crawling speed on smooth agar surface, (ii) thrashing frequency in liquid media, and (ii) burrowing through a soft gel. However, subtle differences cannot be captured through crawling or thrashing assays. The 3-D matrix of the gel structure in the burrowing assay provides greater mechanical resistance, limiting movement to a greater extent and extends the capability of separating subtle defects. We will utilize the strain PJ1046, containing a mutation in the cha-1 gene locus, which encodes a choline acetyltransferase in the neuronal synapses of the worms. Being a temperature dependent mutation, at 25C there will be significant impairment in the locomotive ability of the worms. We can then in effect create samples with varying levels of neuromuscular functioning by incubating them for different amounts of time at 25C. The animals will be suspended at the bottom of a well plate filled with Pluronic F-127 gel and monitored for the amount reaching the top after an elapsed time of 120 minutes. This assay can be used as a means to validate the efficacy for measuring the burrowing of C. elegans in screening neuromuscular functioning.

Xing, Xiaojuan (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Larvae nematode C.elegans evaluate neurobehavioral toxicity to metallic salts"

Xing, Xiaojuan

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In this study, we investigated the locomotion behavior changes at different developmental stages in Caenorhabditis elegans exposed to metals for 4h. No obvious differences could be observed in young adults exposed to examined metals, and only exposure to 100 mM of examined metals could signi?cantly decrease the locomotion behaviors of L4 larvae. In contrast, exposure to 50 and 100 mM of examined metals induced noticeable repression of locomotion behaviors at L1L3 larval stages, and a signi?cant decrease of locomotion behaviors could be observed in L1 larvae exposed to Pb and Hg, and in L2 larvae exposed to Hg at the concentration of 2.5 mM. Moreover, the L1-, L2-, and L3- larvae exposed to metals for 4h exhibited similar neuro behavioral toxicity manner to L4- larvae exposed to metals for 24h. Therefore, younger larvae showed more severe de?cits in neuro behavioral phenotypes than L 4 larvae and young adults in metal-exposed nematodes.

Nicholas J D Gower et al. (2001) International C. elegans Meeting "Dissection of the upstream regulatory elements of the Inositol 1,4,5-trisphosphate receptor gene itr-1"

Gillian R Temple, Jacqueline E Schein, Howard A Baylis, Denise S Walker, Marco Marra, Nicholas J D Gower

[International C. elegans Meeting, 2001]

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a central role in the organisation of calcium signals by regulating intracellular calcium flux. IP3Rs in C. elegans are encoded by a single gene, itr-1 (also called lfe-1 (1) and dec-4 (2) ). In previous work (3), we characterised the gene structure in detail and identified three putative transcriptional start sites and three alternative splice sites. Sequence comparisons to the C. briggsae itr-1 homologue support the hypothesis that the itr-1 gene has three promoters (pA, pB and pC), each of which gives rise to an alternative mRNA and hence unique protein. The pattern of expression directed by each promoter was determined using GFP reporter gene fusions. pA directs expression in the pharyngeal terminal bulb, the rectal epithelial cells and vulva; pB directs expression in the amphid socket cells, the PDA motor neuron and the spermatheca; pC directs expression in the spermathecal valve, uterine sheath cells, pharyngeal isthmus and intestine (4). Thus tissue-specific expression of itr-1 variants is directed by three promoters and this results in adjacent cells in the same tissue containing different IP3R isoforms. Analysis of the sequence conversation between promoter A of C. elegans and C. briggsae identified four short regions (pA-A to pA-D) of conservation. Deletion analysis of pA demonstrated that the region containing pA-C is required for expression in the terminal bulb and rectal epithelial cells and the region containing pA-D is required for expression in the vulva. pA-C contains sequences which show homology to binding sites for known C. elegans transcription factors. We are currently identifying factors that regulate the promoter using a yeast one-hybrid screen. 1. Clandinin et al (1998) Cell: 92, 523-533. 2. Dal Santo et al (1999) Cell: 98 757-767. 3. Baylis et al (1999) J. Mol Biol: 294, 467-476. 4. Gower et al (2001) J. Mol Biol: 306, 145-157.

Baritaud, Mathieu et al. (2015) International Worm Meeting "Targeting mitochondria in muscular dystrophies through acting on ROS, mitohormesis and programmed cell death pathways ."

Kretz-Remy, Carole, Martin, Edwige, Gieseler, Kathrin, Baritaud, Mathieu, Pierson, Laura, Walter, Ludivine, Mariol, Marie-Christine, Chazaud, Benedicte

[International Worm Meeting, 2015]

Muscular dystrophies are characterized by muscle weakness and degeneration induced by genetic mutation of muscle structure or signaling components. The development of treatments against dystrophies is challenging, in part due to various primary genetic defects affecting different cellular functions. Nonetheless, many studies observed mitochondrial dysfonctions in muscle pathologies. Moreover, using a worm model of Duchenne Muscular Dystrophy (DMD) our team clearly demonstrated an implication of the mitochondrial key effectors Dynamin Related Protein-1, (DRP-1) and cytochrome c in the early process of muscle degeneration (MD). We are now focusing on Reactive Oxygen Species (ROS) and Programmed Cell Death (PCD) pathways to identify common mitochondrial disorders that contribute to MD in muscular dystrophies. We established different C. elegans models that mimic human muscular dystrophies such as Duchenne and Becker dystrophies, Limb-Girdle, Emery-Dreifuss or congenital muscular dystrophy. We revealed in the C. elegans model for DMD that MD can be reduced by treatments with the mitohormetic compound Paraquat (0,1mM) or the antioxidant compounds NAC and vitamin C. This observation suggested that ROS pathways mediated by mitochondria could be targets to reduce MD. In addition, our preliminary results indicate that these treatments are also efficient against MD in other C. elegans models for human muscular dystrophies. Moreover, using a gain of function mutation in the anti-apoptotic effector ced-9 gene (ortholog of Bcl-2) and RNA interference of key molecular actors of PCD linked to mitochondria: ced-3, ced-4, ced-13 and egl-1, we investigated whether specific mitochondrial PCD pathways are involved in MD. To validate our data obtained in C. elegans, we use human immortalized myoblasts from healthy or DMD patients. Preliminary data indicated that DMD myoblasts proliferate less and have a reduced fusion capacity during myogenic differentiation than myoblasts from healthy patients. Ongoing investigations examine mitochondrial pathways identified in C. elegans by challenging myoblasts for proliferation, cell fusion and oxidative stress sensibility.Our original approach using both worm models and human myoblasts, will allow us to establish whether manipulating mitochondrial pathways, particularly ROS signaling and PCD pathways can reduce progression of MD in muscular dystrophies.

zhou, shan et al. (2009) International Worm Meeting "STN-2/g-syntrophin mediates SAX-7/L1CAM function in maintaining neuronal positioning by linking SAX-7 to DYS-1/dystrophin."

opperman, karla, chen, lihsia, zhou, shan

[International Worm Meeting, 2009]

Cell adhesion receptors are essential in multiple processes that range from cell migration to maintaining tissue integrity. As transmembrane proteins, cell adhesion molecules (CAMs) convert extracellular signals into cellular responses via cross-talk with cytoskeletal and intracellular signaling networks. The mechanisms by which CAMs relay these signals are still not clear, particularly with those belonging to the immunoglobulin (Ig) superfamily. L1CAMs are single-pass transmembrane IgCAMs that are conserved in metazoans. Mammals have four L1CAMs (L1, NrCAM, CHL1, and Neurofascin) that are required for nervous system functions. Polymorphisms in NrCAM and CHL1 are associated with autism and schizophrenia while mutations in L1 cause the neurological CRASH disorder, the symptoms of which include mental retardation and spastic paraplegia. The roles of L1CAMs underlying these diseases are not clear. C. elegans has two L1CAMs: SAX-7 and LAD-2, which is a non-canonical L1CAM with a divergent cytoplasmic tail. We previously showed SAX-7 and LAD-2 have non-overlapping functions with LAD-2 mediating axon guidance and SAX-7 maintaining the positions of motor neurons along the ventral nerve cord. We determined that for proper function, SAX-7 requires intracellular interactions that include STN-2/g-syntrophin. Syntrophins are known to associate with dystrophin, an actin-binding molecule that is best characterized for its role in maintaining muscle integrity. Mutations in dystrophin result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. In addition to progressive muscle weakness, many DMD patients also exhibit cognitive deficits, including mental retardation, thus revealing as-yet-unidentified neuronal roles for dystrophin. We hypothesize that STN-2/g-syntrophin mediates SAX-7 function in maintaining neuron positions by linking SAX-7 to dystrophin; linkage to dystrophin likely provides SAX-7 anchorage to the actin cytoskeleton, thus leading to increased SAX-7-mediated cell adhesion. Consistent with the hypothesis, our analysis revealed that dys-1 genetically interacts with sax-7 and plays a role in maintaining neuronal positioning. Furthermore, our protein recruitment assays in HEK293 cells strongly support a molecular interaction between SAX-7 and DYS-1 that requires the presence of STN-2 as a linking molecule. Lastly, we show that STN-2 is required in neurons to mediate SAX-7 function in maintaining neuronal positioning. Taken together, these results point to a novel role for dystrophin in maintaining neuronal positions via its molecular interaction with L1CAMs.

Huayue Ye et al. (2008) "Molecular control of the retrieval and prevention functions in stress-induced learning defects from vitamin E administration in Caenorhabditis elegans"

Huayue Ye, Dayong Wang

[2008]

"Vitamin E (α-tocopherol), a potent radical chain breaking antioxidant, is popularly used as ancillary drug for neurodegeneration disease, oxidative stress-related impairment and cognitive dysfunction. In the current work, we explored the thermosensation model to investigate the effects of vitamin E administration on learning behaviors and the related molecular mechanism in Caenorhabditis elegans. In the assay model, administration of 100 μg/mL and 200 μg/mL vitamin E had no significant effects on learning behaviors, whereas relatively high concentration (400 μg/mL) of vitamin E exposure shortened the acquisition period of the association paradigm. Pre-treatment or post-treatment with 200 μg/mL vitamin E could prevent or retrieve and even enhance the UV irradiation- and heavy metal (Al and Pb) exposure- induced learning defects. Moreover, treatment with 200 μg/mL vitamin E could restore the learning formed in the ncs-1 and hen-1 mutant worms, suggesting that vitamin E can largely mimic the function of NCS-1 and HEN-1 in regulating the learning behaviors. In addition, vitamin E could also mimic the function of DEG-1, which plays a key role in the regulation of neurodegeneration, and MEV-1, whose mutation will result an altered sensitivity to oxidative stress. Therefore, our data suggest that the direct association may exist between trace dietary vitamin E intake and learning impairment induced by oxidative stress, and a specific response mechanism may be activated or even be amplified after the administration of vitamin E. Moreover, trace vitamin E administration may ameliorate the metal exposure- and/or the UV irradiation-induced learning impairment at least partially by regulating the NCS-1 functions in learning control in C. elegans."

K Wah Chu et al. (2001) International C. elegans Meeting "Synergistic toxicity of multiple heavy metals is revealed by a biological assay using C. elegans and its transgenic derivatives."

King L Chow, K Wah Chu

[International C. elegans Meeting, 2001]

Caenorhabditis elegans with a short life cycle, low maintenance cost, high tolerance to pH, salinity, water hardness and temperature as well as its well-characterized genetics makes it an reliable model as bioindicator for monitoring effluent or water samples from freshwater or estuarine sources. It eliminates the problem of heterogeneous population occurred in other field collected species used in bioassay. We fine-tuned the toxicity assay with the use of L1 larvae. Toxicity of 12 different metal ions have been tested by exposing C. elegans and its transgenic derivatives to individual metals. Based on the LC 50 value in these tests, we showed that the toxicity of these metals could be ranked as Hg> Cu> Pb> Cr> Cd> Al> Co> Ni> Zn> Mn. We further demonstrated that there were combinatorial effects of these heavy metals on the survival of C. elegans . Both synergistic and rescue effect could be observed in the paired-metal toxicity bioassay. Mixed doses of two metals at concentrations with weak lethal effect could lead to massive killing of animals. Copper, coupled with other metals, elicits the strongest synergistic results, while zinc could rescue the toxic impact of other metals on C. elegans . Our observation indicates that the current practice of regular monitoring of heavy metal contents in environmental samples by physical or chemical means might not be effective in determining the actual toxicity, where bioavailablity and combinatorial effect are neglected. In order to improve the sensitivity of our assay, we had generated and tested multiple transgenic strains carrying a heat shock promoter driving gfp reporter in substitution of wild type worms. The duration of the assay could be reduced about 10 folds and the assay completed in hours, while synergistic effect between toxicants could still be revealed. With further optimization, regular year round environmental monitoring with these transgenic strains should be possible.

Olena Vatamaniuk et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Biochemical Interactions And Genetic Analyses Of ABC Transporters Required For Heavy Metal Detoxification"

Olena Vatamaniuk, Andy Chen, Devarshi Selote

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Understanding the cellular mechanisms of heavy metal (e.g. cadmium [Cd], mercury [Hg] and lead [Pb]) detoxification is critical for the cure and prevention of heavy metal-caused diseases, such as neurodegenerative conditions, dysfunction of vital organs, and cancer. Among the major contributors to heavy metal detoxification are ATP-binding cassette (ABC) transporters. The specific family members involved, and their functions in evolutionarily diverse organisms, however, are not understood. The recently discovered acute requirement of a half-molecule ABC transporter, CeHMT-1, in Cd detoxification in the nematode worm, Caenorhabditis elegans, provides a powerful approach to study the mechanisms, structural requirements, and components of the CeHMT-1 metal detoxification pathway. Unique structural features of CeHMT-1 (it is the only half molecule ABC protein in C. elegans possessing the hydrophobic N-terminal extension (NTE)) define the HMT-1 subfamily in different species, including humans. Although it is suggested that half molecule ABC transporters must associate with other family members and/or other cellular proteins, how HMT-1 proteins detoxify heavy metals and how this subfamily of ABC transporters functions is unknown. To understand the mechanism of the CeHMT-1 functional activity, we study its genetic and biochemical interactions, its tissue-specific expression and subcellular localization. Towards this goal we have establishes that CeHMT-1 forms higher oligomeric complex and localizes to the lysosomal-like compartment in intestinal cells. To identify putative interacting partners of CeHMT-1, we have screened 30 from 60 ABC transporters in C. elegans genome and identified six ABC transporters in addition to CeHMT-1 that are involved in metal detoxification. Their role as compared to CeHMT-1 in providing heavy metal tolerance will be discussed. In addition, we will present data demonstrating that our screens have the potential to identify new pathways, and to ascribe functions to known pathways, not previously determined to be involved in heavy metal tolerance and, thus, will contribute to our understanding of cellular resources for metal detoxification.