Ellwood, R.A. et al. (2019) International Worm Meeting "Prednisone improves mitochondrial function but not excitation contraction coupling in dys-1."

Piasecki, M., Vanapalli, S.A., Szewczyk, N., Hewitt, J.E., Deane, C.S., Etheridge, T., Ellwood, R.A.

[International Worm Meeting, 2019]

We recently found that prednisone, the current standard treatment for Duchenne muscular dystrophy (DMD), improves thrashing rate, muscle strength, and mitochondrial respiration in dys-1 worms (Hewitt, et al., 2018). Given that prednisone is an anti-inflammatory agent and that C. elegans largely lack a mammalian inflammatory system, we were interested in better understanding how prednisone works. Since excitation contraction coupling is required for normal muscle function, we tested prednisone's effect on the dys-1's levamisole resistance and surprisingly found little improvement. This suggests that prednisone is not improving the altered calcium dynamics in dys-1. Since dys-1 animals are fainters, another phenotype associated with altered calcium dynamics, we tested if dys-1 animals are halothane hypersensitive. Unsurprisingly, we found that dys-1 animals are hypersensitive to halothane and that this hypersensitivity is not reversed by prednisone. Together, these results raise the question of if dys-1 halothane sensitivity is related to unc-68 malignant hypothermia variant's halothane sensitivity. These results also suggest that future drug discovery studies should target the excitation contraction coupling defect in dys-1. Finding that prednisone did not substantially improve calcium handling, we hypothesized that prednisone improved muscle health by increasing mitochondrial biogenesis and/or altering mitochondrial function during the major expansion of mitochondria in the later larval stages. Surprisingly, we found treatment of fully developed adult dys-1 worms with prednisone both increased thrashing rates and delayed declines in thrashing with age compared to untreated controls. This result suggests a more detailed analysis of prednisone's acute effect on mitochondrial function is required, which could lead to discovery of compounds that do not have the same undesired side effects as prednisone.

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.

Musset-Bilal F et al. (1996) East Coast Worm Meeting "Structural and functional similarities between mammalian and C. elegans SPARC proteins"

Musset-Bilal F, Schwarzbauer JE, Fitzgerald MC

[East Coast Worm Meeting, 1996]

The basement membranes of nematodes and vertebrates are composed of similar protein components including laminin, perlecan, type IV collagen, and SPARC. The sequence conservation of these proteins suggests that basement membrane structure and function are conserved. To determine the extent of the structural similarities between C. elegans and vertebrate SPARC proteins, we have raised polyclonal antibodies against a bacterial fusion protein containing one of the calcium binding domains of nematode SPARC. Immunoblots of whole nematode lysates and of NP40 soluble proteins showed that nematode SPARC is a 27 kD protein (1). When electrophoresed under nonreducing conditions, SPARC shows increased mobility demonstrating that it has compact, disulfidebonded structure. Similarly, treatment with N-glycosidase F, which removes complex carbohydrates, resulted in increased mobility. Therefore, SPARC is glycosylated, probably on the conserved asparagine residue in the cysteine-rich domain. Together these biochemical analyses show that nematode SPARC is structurally very similar to its vertebrate counterparts. In mammalian cell culture, excess SPARC protein has been shown to reverse cell adhesion and cause cell rounding. Overexpression of wild type SPARC in nematodes causes body deformities and paralysis which could result from defects in muscle cell adhesion (2). To determine the effects of SPARC on muscle cell structure, a transgenic line was generated that carries extra copies of the SPARC gene (ost-l) along with the rol-6(d) gene N2;Ex[ost-1;rol-6(d)]). L4 larvae from this line develop an Unc phenotype with a protruding vulva, paralysis, and sterility. These animals were stained with fluorescently labeled phalloidin to visualize actin filament organization. While wild type animals form distinct sex muscle cell structures that attach at the vulva, the N2;Ex[ost-l;rol-6(d)] animals show no actin organization in their sex muscle cells. We conclude that the vulval protrusion observed in the presence of excess SPARC results from improper cytoskeletal organization of the sex muscle cells. As actin filament structure is dependent on cell adhesion, these results support the idea that in these transgenic animals SPARC is disrupting muscle cell adhesion. 1. Schwarzbauer, J.E., F. MussetBilal, and C.S. Ryan (1994) The extracellular matrix associated protein SPARC/osteonectin in Caenorhabditis elegans. Meth. Enzymol. 245, 257-270. 2. Schwarzbauer, J.E. and C.S. Spencer (1993) The Caenorhabditis elegans homolog of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility. Mol. Biol. Cell 4, 941- 952.

Rui Wang et al. (2007) International Worm Meeting "The TARP-like proteins, STG-1 and STG-2, differentially affect signaling via the GLR-1 glutamate receptor subunit in C. elegans."

Penelope J. Brockie, Andres V. Maricq, Jerry E. Mellem, David M. Madsen, Craig S. Walker, Michael M. Francis, Rui Wang

[International Worm Meeting, 2007]

Understanding how functional nervous systems are established and maintained is a fundamental problem in neuroscience. Glutamate is an essential neurotransmitter found in all well-characterized nervous systems and is used to transmit excitatory information at synapses. There are at least three classes of ionotropic glutamate receptors (iGluRs), AMPA, kainate and NMDA that mediate rapid glutamatergic signaling. In vertebrates, members of the AMPA class are of particular interest because of their relevance to neurological disorders and cellular models of learning. We would like to understand the molecular mechanisms that regulate the function of this important class of iGluRs. Using genetic, behavioral and electrophysiological analyses we have begun to unravel the molecular requirements of functional glutamatergic synapses. In vertebrates, the trafficking, localization and function of AMPA receptors are partially dependent on a family of Transmembrane AMPA receptor Regulatory Proteins (TARPs), but little is known about how TARPs regulate synaptic function and their relevance to behavior. The C. elegans</I> AMPA receptor subunit GLR-1 requires two auxiliary subunits for function, SOL-1 (a CUB-domain transmembrane protein) and the TARP-like protein STG-1 (1, 2). Using knockout techniques and sensitized genetic screens, we have now identified a second TARP-like protein, STG-2, that is essential for glutamatergic signaling in the worm. We have shown that STG-1 and STG-2 modify GLR-1 function, are differentially expressed in neurons, and help tailor glutamatergic neurotransmission to specific avoidance behaviors. Similar to the loss of SOL-1, mutations in both stg-1</I> and stg-2</I> result in behavioral and electrophysiological defects that phenocopy the loss of GLR-1. Interestingly, mutating either stg-1</I> or stg-2</I> alone results in different phenotypes suggesting that STG-1 and STG-2 have both overlapping and distinct roles in regulating AMPA receptor function and thus worm behavior. 1. Zheng, Y., et al. (2004). Nature</I> 427: 451-7 2. Walker, C.S., et al. (2006). PNAS</I> 103(28): 10781-6.

Schumacher JM et al. (1997) Worm Breeder's Gazette "AIR-1 and AIR-2: Two highly related, conserved, and uniquely localized serine/threonine protein kinases that are both required for the proper execution of cell cycle events in C. elegans embryos"

Donovan PJ, Golden A, Schumacher JM

[Worm Breeder's Gazette, 1997]

A number of laboratories have recently described various members of a highly conserved serine/threonine protein kinase family that appears to play a role in chromosome segregation and mitotic spindle dynamics in several different organisms (1,2,3,4). A search of the C. elegans genome database revealed that there are at least two nematode proteins that are highly related to this protein kinase family. We refer to these proteins as AIR-1 and AIR-2 (Aurora/Ipl-1 related). To date, the subcellular location of only one member of this protein kinase family has been reported (3,4). The mammalian protein Iak-1 has been shown to be associated with the centrosomes of the mitotic spindle in tissue culture cells and with meiotic chromosomes in mouse spermatocytes (J. Schumacher, unpublished). By performing immunocytochemistry experiments on fixed C. elegans embryos with antisera raised against specific peptides, we have found that the AIR-1 protein, like its mammalian counterpart, is also found on centrosomes in mitotic cells. The protein is first detectable on centrosomes of the first mitotic division following pronuclear fusion in the one-cell embryo. It is clearly associated with duplicated centrosomes prior to their migration to opposite sides of the nucleus and is found on mitotic centrosomes up to the limits of resolution in two-fold stage embryos. The location of the AIR-2 protein mimics that of Iak-1 in meiotic cells. Like Iak-1 in spermatocytes, AIR-2 is also found on chromosomes undergoing meiotic divisions both in oocytes and spermatocytes. AIR-2 staining is diffuse throughout the cellularized oocytes of the proximal gonad, but becomes localized to the chromosomes in the oocyte that resides next to the spermatheca. The protein persists on these chromosomes throughout meiosis and remains associated with polar body chromatin following these divisions. AIR-2 is also found on meiotic chromosomes during spermatogenesis in C. elegans males. In addition, it is associated with mature sperm present in the spermatheca, but at this stage it doesn!t appear to be localized to the chromatin. Instead, it appears to surround the sperm, suggesting an association with the cellular membrane. In embryos, diffuse AIR-2 staining is found in the cytoplasm, but is also clearly localized to mitotic metaphase chromosomes. The protein may be present on chromosomes at other stages of mitosis, but is difficult to detect on less condensed chromatin. By telophase, AIR-2 is clearly localized to midbody microtubules, and a small dot of staining persists on the cell membrane once cytokinesis is complete. To disrupt the function of each of these proteins during embryogenesis, we injected antisense RNA corresponding to the entire cDNA of each gene into the gonads of C. elegans hermaphrodites. Injection of either RNA resulted in embryonic lethality and the specific loss of each protein as detected by immunocytochemistry. AIR-1 deficient embryos die with greater than 100 cells and are severely aneuploid. Analysis of younger embryos revealed a variety of chromosome segregation defects ranging from the loss of a single chromosome to the missegregation of every chromosome to one daughter cell in a particular division. Some cells also appeared to be severely polyploid and contain multiple centrosomes, suggesting multiple cell cycles that lack an intervening mitotic division. Disruption of AIR-2 resulted in the production of one-cell embryos that contained many nuclei and centrosomes, as well as polar bodies that continue to replicate and divide. One-cell embryos containing anywhere from one to greater than 20 nuclei were found with equally abnormal numbers of centrosomes. This dramatic phenotype again suggests the uncoupling of DNA replication and centrosome duplication from the completion of mitosis. References: 1) Chan, C.S. and Botstein, D. (1993). Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast. Genetics 135, 677-691 2) Glover, D. M., Leibowitz, M. H., McLean, D. A., and Parry, H. (1995). Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81, 95-105. 3) Gopalan, G., Chan C.S., and Donovan P.J. (1997). A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators. J Cell Biol 138, 643-656 4) Kimura, M., Kotani, S., Hattori, T., Sumi, N., Yoshioka, T., Todokoro, K., and Okano, Y. Cell cycle-dependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1. J Biol Chem 272, 13766-13771 Research sponsored by the National Cancer Institute, DHHS, under contract with ABL.