Gaffney CJ et al. (2014) J Vis Exp "Methods to assess subcellular compartments of muscle in C. elegans."

Barratt TF, Gaffney CJ, Szewczyk NJ, Bass JJ

[J Vis Exp, 2014]

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm's body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Gaffney CJ et al. (2018) Aging (Albany NY) "Greater loss of mitochondrial function with ageing is associated with earlier onset of sarcopenia in C. elegans."

Constantin-Teodosiu D, Pollard A, Barratt TF, Szewczyk NJ, Greenhaff PL, Gaffney CJ

[Aging (Albany NY), 2018]

Sarcopenia, the age-related decline of muscle, is a significant and growing public health burden. <i>C. elegans</i>, a model organism for investigating the mechanisms of ageing, also displays sarcopenia, but the underlying mechanism(s) remain elusive. Here, we use <i>C. elegans</i> natural scaling of lifespan in response to temperature to examine the relationship between mitochondrial content, mitochondrial function, and sarcopenia. Mitochondrial content and maximal mitochondrial ATP production rates (MAPR) display an inverse relationship to lifespan, while onset of MAPR decline displays a direct relationship. Muscle mitochondrial structure, sarcomere structure, and movement decline also display a direct relationship with longevity. Notably, the decline in mitochondrial network structure occurs earlier than sarcomere decline, and correlates more strongly with loss of movement, and scales with lifespan. These results suggest that mitochondrial function is critical in the ageing process and more robustly explains the onset and progression of sarcopenia than loss of sarcomere structure.

Gaffney CJ et al. (2016) J Cachexia Sarcopenia Muscle "Degenerin channel activation causes caspase-mediated protein degradation and mitochondrial dysfunction in adult C.elegans muscle."

Rose A, Chu J, Gaffney CJ, Constantin-Teodosiu D, Baillie DL, Shephard F, Szewczyk NJ, Greenhaff PL

[J Cachexia Sarcopenia Muscle, 2016]

BACKGROUND: Declines in skeletal muscle structure and function are found in various clinical populations, but the intramuscular proteolytic pathways that govern declines in these individuals remain relatively poorly understood. The nematode Caenorhabditis elegans has been developed into a model for identifying and understanding these pathways. Recently, it was reported that UNC-105/degenerin channel activation produced muscle protein degradation via an unknown mechanism. METHODS: Generation of transgenic and double mutant C.elegans, RNAi, and drug treatments were utilized to assess molecular events governing protein degradation. Western blots were used to measure protein content. Cationic dyes and adenosine triphosphate (ATP) production assays were utilized to measure mitochondrial function. RESULTS: unc-105 gain-of-function mutants display aberrant muscle protein degradation and a movement defect; both are reduced in intragenic revertants and in let-2 mutants that gate the hyperactive UNC-105 channel. Degradation is not suppressed by interventions suppressing proteasome-mediated, autophagy-mediated, or calpain-mediated degradation nor by suppressors of degenerin-induced neurodegeneration. Protein degradation, but not the movement defect, is decreased by treatment with caspase inhibitors or RNAi against ced-3 or ced-4. Adult unc-105 muscles display a time-dependent fragmentation of the mitochondrial reticulum that is associated with impaired mitochondrial membrane potential and that correlates with decreased rates of maximal ATP production. Reduced levels of CED-4, which is sufficient to activate CED-3 in vitro, are observed in unc-105 mitochondrial isolations. CONCLUSIONS: Constitutive cationic influx into muscle appears to cause caspase degradation of cytosolic proteins as the result of mitochondrial dysfunction, which may be relevant to ageing and sarcopenia.

Denham DA et al. (1989) J Helminthol "Experimental Brugia pahangi and B. malayi infections of callitrichid primates."

Suswillo RR, Hetherington CM, Denham DA

[J Helminthol, 1989]

The callitrichid primates, Callithrix jacchus jacchus (the marmoset) and Saguinus labiatus (the tamarin) were inoculated with infective larvae of Brugia malayi and B. pahangi. Microfilaraemia at low levels developed in 3 out of 4 C.j. jacchus infected with B. malayi and living or dead adult worms found in all 4. Only one of 4 C.j. jacchus became microfilaraemic (mf + ve) when given B. pahangi and adults were found in two. Of 4 S. labiatus given B. pahangi one became very lightly mf + ve and adults were found in 3. It is concluded that these animals are not suitable hosts for chemotherapeutic experiments.

Pollard, A.K. et al. (2017) International Worm Meeting "Worms in space for outreach on Earth."

Gaffney, C.J., Szewczyk, N.J., Pollard, A.K., Phillips, B.E., Etheridge, T.

[International Worm Meeting, 2017]

Muscle atrophy is one of the main (mal)adaptations associated with prolonged spaceflight. We have previously established C. elegans as a model for studying spaceflight induced alterations in muscle with past experiments demonstrating reproducible expression changes in cytoskeletal and metabolic proteins and their encoding mRNAs. We are currently preparing a European Space Agency sponsored spaceflight experiment that aims to manipulate the stability of integrin based muscle attachment complexes and insulin-like signalling to determine if either or both of these predicted molecular mechanisms causally regulate the gene and protein expression changes observed in response to spaceflight. As spaceflight, particularly launches, captures the public's imagination, we have planned outreach activities to capitalize on this existing interest in spaceflight and to raise the profile of space-based life science research in the United Kingdom (UK). We have divided our outreach messages into three main themes. Firstly, we will describe the advantages of using C. elegans as a model to study biology and highlight some of the impacts on human health that have arisen from studies of the worm. Secondly, we will discuss the use of C. elegans as a model to study space physiology with a focus on how our past and current experiments were physically conducted. Thirdly, we will raise awareness of how the findings from our studies are being translated into new knowledge of the regulation of human muscle both on Earth and in space. We will present examples of how each message will be presented in a poster format for use at our outreach activities and provide electronic versions of these posters for other educators who may wish to use them. Three distinct dissemination routes will be used to maximise the impact of our outreach. Firstly, we will engage with the UK media to produce a video documentary of our experiment to provide the public with deeper understanding of how space experiments are actually conducted. Secondly, outreach days will be run at museums in each of the capital cities in the UK. Thirdly, we will run a number of hands-on workshops at schools in the middle and South West of England. For the outreach days we will employ the three posters described above and an extended version of our school workshop activities. The workshop activities will cover all three of our outreach messages, using wild-type worms and those with mutations relevant to our spaceflight studies (e.g., unc-112 and cha-1), and giving students the opportunity to handle spaceflight experimental hardware. We will discuss the details of our workshop activities for other educators who wish to run similar activities.

Hewitt, J.E. et al. (2019) International Worm Meeting "Robust phenotyping of a C. elegans model of muscular dystrophy reveals clinically relevant phenotypes and drug responses."

Vanapalli, S.A., Gaffney, C.J., Etheridge, T., Pollard, A.K., Lesanpezeshki, L., Hewitt, J.E., Deane, C.S., Szewczyk, N.J.

[International Worm Meeting, 2019]

The most common type of muscular dystrophy, Duchenne muscular dystrophy (DMD), affects 1 of every 3500 live male births. Currently, the disease is poorly understood and there is no cure-only treatments such as prednisone to control symptoms. The disease is caused by a mutation in the dystrophin gene; The C. elegans homologous gene, dys-1, gives the opportunity for testing compounds and improving understanding of mechanisms of DMD in worms. To facilitate this work we have been examining new phenotypes of dys-1 and their response to drug treatment. For this study, we utilized dys-1(eg33), which is reported as having a nonsense mutation. Using our NemaFlex platform we found dys-1 animals were significantly weaker than their wild-type counterparts and were also deficient in thrashing. We also find that dys-1 mutants display severe mitochondrial fragmentation, levamisole resistance, depolarization of the mitochondrial membrane, and an abnormally high basal oxygen consumption rate (OCR). We then tested the effects of prednisone and melatonin on dys-1 mutants and found an improvement in muscle strength, thrashing rate, and mitochondrial network integrity under drug treatments. Prednisone also returns basal OCR to wild-type-like levels, but it does not correct the depolarization of the mitochondrial membrane. We are currently phenotyping the impact of treatment with prednisone, melatonin, and serotonin by assaying in three unique mechanical environments: crawling (via NemaFlex), swimming (via C. elegans Swim Test Software - CeleST), and burrowing (via a novel, hydrogel burrowing assay). Using this platform, we have found that it is possible to uncover phenotypes that are not corrected by drug treatment, suggesting that our multi-environment phenotyping is of value both for drug discovery and mechanistic work with dys-1.

Kravtsov, V. et al. (2013) International Worm Meeting "The effects of aging on dendritic plasticity and PVD-FLP branch coexistence."

Podbilewicz, B., Oren-Suissa, M., Kravtsov, V.

[International Worm Meeting, 2013]

Self-avoidance, tiling and coexistence are the main mechanisms that enable the best dendritic coverage. C. elegans undergoes aging-associated changes that ultimately lead to decreased functionality of the organism, including its neurological functions. Recent research has shown that the nervous system of C. elegans undergoes changes and alterations during aging including dendritic morphology (Tank et al., 2011). We study dendritic plasticity, aging and spatial dendritic organization of two highly arborized mechanoreceptors in C. elegans, PVD and FLP (Oren-Suissa et al., 2010). PVD dendrites of L4s and young adults show regenerative ability following dendrotomy (laser induced severing of dendrites). Our working hypothesis is that in older ages this ability to regenerate is compromised. Previous studies and our preliminary results indicate that PVD and FLP do not overlap in larval stages (Smith et al., 2010). In addition, dendrites within each bilateral PVD do not overlap through a self-avoidance mechanism (Smith et al., 2012). We found that (1) the coverage fields of the PVD and FLP overlap in adult worms, which indicates coexistence and not tiling. This overlap increases as the worm ages. (2) PVDs show aberrant arborization at the age of 9 days of adulthood. (3) Dramatic increase in self-avoidance defects as animals age. In humans many neurodegenerative diseases as well as generalized cognitive decline are associated with age, aberrant arborization or both (e.g. autism and Alzheimer's disease). However our understanding of how these disorders are triggered and aggravated is scarce. Our research provides an insight into the aging and regeneration process of individual neurons. Oren-Suissa, M., et al. (2010). Science 328, 1285-1288. Smith, C.J. et al. (2010). Developmental Biology 345, 18-33. Smith, C.J., et al. (2012). Nature Neuroscience 15, 731-737. Tank, E.M.H. et al. (2011). Journal of Neuroscience 31, 9279-9288.

McCormick, Allyson V. et al. (2009) International Worm Meeting "Characterization of seizure modifiers in worms lacking CaMKII function."

McCormick, Allyson V., Kraemer, Brian, Thomas, James H.

[International Worm Meeting, 2009]

Several C. elegans mutants have seizures with acute exposure to neurostimulants1,2 (e.g. pentylenetetrazole). Loss-of-function alleles of unc-43, the worm homologue of CaMKII, have robust full-body convulsions easily induced by elevated temperature and drug exposure. To further describe worm seizures genetically, we have conducted pilot EMS screens for suppressors and enhancers of unc-43 seizures. Thus far, we have isolated fourteen suppressors and enhancers. Since unc-43(lf) animals have a range of phenotypes caused by a lack of function in specific tissues, modifiers have been classified by changes in seizures and a variety of other behaviors (e.g. spontaneous reversal rate). Characterization of genetic lesions is currently underway. Though identification can be time consuming, a key advantage of forward genetic screens is to isolate single point mutations that create more subtle variations in gene function. This is exciting since so little is known about modifiers in human epilepsies. Such modifiers may contribute to drug resistance, seizure frequency and developmental timing. 1Williams, S.N. et al. (2004). Hum Mol Genet (13) 2043-59. 2Locke, C.J. et al. (2006). Brains Res (1120) 23-34. .

Hannak E et al. (2002) J Cell Biol "The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent."

Habermann B, Hannak E, Kirkham M, Gonczy P, Oegema K, Hyman AA

[J Cell Biol, 2002]

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.

Daniel L Chase et al. (2003) International Worm Meeting "Dopamine-resistant mutants of C. elegans identify components of Go and Gq signaling pathways"

Daniel L Chase, Judy S Pepper, Michael R Koelle

[International Worm Meeting, 2003]

Dopamine, a major neurotransmitter in the mammalian central nervous system, acts through two classes of G protein-coupled receptors known as D1-like and D2-like receptors. These two classes of receptor signal to antagonistically regulate neural activity. Despite intensive research, the signaling mechanisms responsible for the antagonistic effects of dopamine in the brain remain debated. We describe the first genetic screen designed to identify proteins required for dopamine signaling. C. elegans uses endogenous dopamine to inhibit locomotion behavior1, and treatment with excess exogenous dopamine paralyzes worms2. We isolated mutants unresponsive to exogenous dopamine and found that they are also defective for endogenous dopamine signaling. Eleven mutations identified five different genes. Four of these genes encode components of the Go and Gq signaling pathways, including the G protein Go itself and subunits of the RGS complex that inhibits Gq signaling. The Go and Gq signaling pathways act antagonistically in C. elegans to control behavior and their components are conserved in humans. Mutations in these signaling components that decrease Go signaling or increase Gq signaling cause dopamine resistance, while mutations that have opposite effects on these two signaling pathways cause dopamine hypersensitivity. Thus the physiological effects of dopamine are mediated by Go in C. elegans and are antagonized by Gq signaling. Such a dopamine signaling mechanism in mammals could explain many of the opposing effects of D1-like and D2-like receptor activation. Our screen also identified one gene that appears to encode a new Go/Gq signaling component. 1. Sawin, E.R., Ranganathan, R., and Horvitz, H.R. (2000). Neuron 26, 619-631; 2. Schafer, W.R., and Kenyon, C.J. (1995). Nature 375, 73-78.