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

[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.

Roxani Gatsi et al. (2005) International Worm Meeting "Suppressor screens for the dissection of the IP3 signalling pathway in the pharynx"

Roxani Gatsi, Howard Baylis

[International Worm Meeting, 2005]

IP3 mediated calcium signalling in C. elegans determines specificity of cellular responses to extracellular stimuli. Specialized IP3 receptors (IP3Rs), which are located on the endoplasmic reticulum membrane, regulate cytoplasmic calcium concentrations that determine various cellular functions. One of these functions is the up-regulation of pharyngeal pumping in response to food (Walker et al, 2002a). Relatively little is known about the IP3-mediated regulation of the rhythmic pumping of the pharynx. Walter et al (2002b) has shown that interaction between IP3Rs and myosin are required for regulation of pharynx pumping but not for other physiological rythmic functions in C. elegans, indicating the importance of protein interactions for determining specificity. In order to identify additional components of the IP3 signalling pathway in the pharynx we performed genetic suppressor screens using an IP3R mutant (sa73), which shows reduced pharyngeal pumping. We have isolated various mutants that suppress various defects of the sa73 phenotype. The results from the characterization of these mutants, as well as the approach to map the mutations will be presented. Walker DS., et al. (2002a) Mol Biol Cell 13, 1329-1337. Walker DS., et al. (2002b) Curr Biol 12, 951-956

Lambie EJ et al. (1991) International C. elegans Meeting "lag-1 AND lag-2 ARE REQUIRED FOR THE FUNCTIONING OF lin-12 AND glp-l."

Kimble JE, Christensen S, Lambie EJ

[International C. elegans Meeting, 1991]

lin-12 and glp-1 encode homologous transmembrane proteins that probably act as receptors in regulatory cell interactions during development (Yochem and Greenwald, 1987). The absence of zygotic lin- 12 produces a low level of L1 lethality (~10%), whereas zygotic glp-1 is not required for embryonic development. However, lin-12 glp-l double mutants invariably arrest as L1s. We interpret this result as indicating a common essential function of lin-12 and glp-l during mid/late embryogenesis. Iin-12 glp-1 double mutants have several anatomical abnormalities that may result from cell fate transformations, e.g. they are missing several structures (rectum, anus, excretory cell) and have duplications of others (excretory pore). We call this combination of characteristics the Lag phenotype (for lin-12 and glp-l). In an effort to identify genes that are required for the functioning of both lin-12 and glp-l, we have isolated 12 recessive mutations that cause a Lag phenotype. These mutations define two loci, lag-l IV, and lag-2 V. Mutations in lag-2 had been previously isolated by Johnsen and Baillie (let-461), and Tax, Thomas and Horvitz (sel-3). Iag-1 is a newly identified gene. We think that both lag-l and lag-2 are required for postembryonic functions of glp-l and lin-12, since certain alleles can cause postembryonic phenotypes diagnostic of lin-12 and/or glp-l mutations. The larval lethality of Lag animals may be due, in part or whole, to the absence of the excretory cell. Analysis of a temperature sensitive allele of lag-2, q420, indicates that the requirement for lag-2 function occurs during mid-embryogenesis, at approximately the time of birth of the excretory cell. Mosaic analysis using a strain of genotype ncl-1 unc-36 lin-12 glp-1; qDp3 suggests that lin-12 and glp- 1 are required in AB.p (from which the excretory cell is descended) for viability; unc-36 is required in AB.p, and Unc nonLag mosaics are extremely rare. We are currently attempting the molecular isolation of lag-l and lag2 in the expectation that this will lead to a better understanding of how they function in concert with lin-12 and glp-1. E.L. is supported by Damon Runyon-Walter Winchell postdoctoral fellowship DRG-989.

Lambie EJ et al. (1991) International C. elegans Meeting "q425: A STERILE VUL, BUT STILL INTERESTING."

Lambie EJ, Kimble JE

[International C. elegans Meeting, 1991]

During an F2 screen for steriles, we isolated a recessive mutation ( q425 I) that produces an interesting combination of phenotypes. Homozygous q425 hermaphrodites are both sterile and vulvaless. Males are superficially normal, but they have not been tested for mating ability. Both males and hermaphrodites are capable of producing sperm; however, hermaphrodites never produce oocytes. In adult hermaphrodites, the proximal pachytene gerrnline nuclei tend to accumulate in the loop region of the gonad, instead of resuming meiosis and proceeding around the loop. One interpretation of this phenotype is that proximal pachytene nuclei normally receive a signal that induces them to resume meiosis and proceed around the loop into the ventral ovary. Some aspect of this signalling process could be disrupted by q425. To learn more about the possible influence of q425 on intercellular signalling, we examined its effect on vulval induction. Inspection of L3s revealed that q425 homozygotes possess an anchor cell, but the vulval precursor cells appear to divide only once, and then enter the hypodermal syncytium. This phenotype is similar to that of some other vulvaless mutants, let-23, lin-2, lin-3, lin-7, and lin-10 (Ferguson, Sternberg and Horvitz, 1987). q425 also resembles this group of vulvaless mutations in that it is not epistatic to a lin-12(d) mutation: q425; lin-12(nl37d) double mutants are Muv (and sterile). This indicates that q425 does not prevent vulval lineages per se, but may affect the decision to adopt a vulval fate. To further subclassify q425, we examined its epistatic relationship to the synMuv mutations, lin-8(nl l l ) and lin-9(nl l 2). q425; lin-8; lin-9 triple mutants are sterile and vulvaless. Mutations in lin-2, lin-7 and lin-10 are also epistatic to synMuv mutations, but lin-3 mutations are not. Because the multivulva phenotype produced by the synMuv mutations does not depend on the presence of the anchor cell, it is thought that lin- 2, lin-7 and lin-10 (and probably q425) are required for the response to the anchor cell signal, whereas lin-3 is required for its generation. q425 maps to the left arm of chromosome I, nearfog-l. No other vulvaless mutations have been reported for this region, so it is possible that q425 represents a newly identified gene. The pleiotropic effects of q425 suggest that it may disrupt cell-cell interactions in many tissues. In this regard, it is notable that while q425 single mutants are fairly well coordinated, q425 dpy-S(e61) double mutants exhibit an unusual shimmying behavior when induced to move backwards. The basis for this coordination defect is not known, but could involve neuraVmuscular signal transduction or the establishment of proper neuronal connectivity. Experiments are in progress to learn more about the genetics and phenotype of q425. (E.J.L. is supported by Damon Runyon-Walter Winchell postdoctoral fellowship DRG-989.)