Boos, J.R. et al. (2019) International Worm Meeting "MitoNEET as a therapeutic target to treat neural circuit decline in Alzheimer's Disease."

Boos, J.R., Geldenhuys, W.J., Saralkar, P.

[International Worm Meeting, 2019]

MitoNEET (CISD1) is a dimeric iron-sulfur mitochondrial protein located on the outer mitochondrial membrane and is believed to be a key regulator in mitochondrial function. MitoNEET contains a ferredoxin cluster binding site, serving as a potential target for drugs. Previous research has suggested that mitoNEET is protective against the formation of reactive oxidative species (ROS) via the regulation of mitochondrial dysfunction and oxidative stress. In recent years, ROS-induced inflammation as a result from mitochondrial dysfunction and oxidative stress have shown to be additional contributing factors towards the progression of Alzheimer's Disease (AD) in addition to the traditional extraneuronal amyloid plaques and intraneuronal neurofibrillary tangles hypotheses. Our aims of this study are to elucidate the effects of mitoNEET as a therapeutic target throughout the progression of AD using a thiazolidinedione class compound we have synthesized (CI-987) and has shown to bind to the ferredoxin cluster binding site of mitoNEET. To examine the effects of CI-987, we used a transgenic AD strain of C. elegans that expresses the extraneuronal A?42 peptide, CL2355, and its wild-type control, CL2122. Both strains were divided into one of three treatment groups: 1) OP50 alone, 2) OP50+vehicle (DMSO), and 3) OP50+100?M CI-987. We assayed sensorimotor, chemosensory, and learning and memory neural circuit function throughout the lifespan in both strains. In this study, we found that treatment with CI-987 slowed the progression of neural circuit decline in normal aging via the CL2122 strain and also throughout the progression of AD via the CL2355 strain. We observed that treating the CL2355 strain with CI-987 showed to be effective in treating early and middle stages of AD neural circuit decline. Furthermore, CL2355 treated with CL2355 produced neural circuit behaviors similar to the CL2122 group treated with OP50. Unfortunately, during the later stages of AD, the promising effects of CI-987 were eventually not enough to combat the neurodegenerative effects as the disease progressed. While the outcomes of this study have shed light on the role of mitoNEET as a therapeutic target throughout the progression of AD and provided an initial foundation for cross-translational studies treating other animal models with CI-987, the need for developing therapeutics that target the neural circuit deficits during in the later stages of AD are still needed.

Boos JR et al. (2022) Adv Redox Res "Structure and biological evaluation of Caenorhabditis elegans CISD-1/mitoNEET, a KLP-17 tail domain homologue, supports attenuation of paraquat-induced oxidative stress through a p38 MAPK-mediated antioxidant defense response."

Taguchi AT, Taylor SJ, Fedun BL, Jandrain HN, Faber SE, Elad SM, Geldenhuys WJ, Hagiuda E, Kumasaka T, Hasegawa K, Iwasaki T, Boos JR

[Adv Redox Res, 2022]

CISD-1/mitoNEET is an evolutionarily conserved outer mitochondrial membrane [2Fe-2S] protein that regulates mitochondrial function and morphology. The [2Fe-2S] clusters are redox reactive and shown to mediate oxidative stress in vitro and in vivo. However, there is limited research studying CISD-1/mitoNEET mediation of oxidative stress in response to environmental stressors. In this study, we have determined the X-ray crystal structure of Caenorhabditis elegans CISD-1/mitoNEET homologue and evaluated the mechanisms of oxidative stress resistance to the pro-oxidant paraquat in age-synchronized populations by generating C. elegans gain and loss of function CISD-1 models. The structure of the C. elegans CISD-1/mitoNEET soluble domain refined at 1.70-A resolution uniquely shows a reversible disulfide linkage at the homo-dimeric interface and also represents the N-terminal tail domain for dimerization of the cognate kinesin motor protein KLP-17 involved in chromosome segregation dynamics and germline development of the nematode. Moreover, overexpression of CISD-1/mitoNEET in C. elegans has revealed beneficial effects on oxidative stress resistance against paraquat-induced reactive oxygen species generation, corroborated by increased activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade.

Hall, David H. et al. (2009) International Worm Meeting "Electron tomography: a new tool for exploring C. elegans cellular anatomy."

Fisher, Kevin, Nguyen, Ken C.Q., Hall, David H., Crocker, Chris, Derr, KD, Rice, William J., Politi, Kristin A., Gunther, Leslie

[International Worm Meeting, 2009]

C. elegans has been studied intensively using serial section reconstruction for several decades, so that every cell type is now known in considerable detail. Nevertheless, limitations of the serial section technique (notably the poor resolution of detail within the depth of each 50 nm "thin" section) have made it difficult to model the shapes of fine details in many organelles, such as the cristae of a mitochondrion, the canaliculi of the excretory canal, membrane ruffles in many cell types, or the constituents at a chemical synapse. High pressure freeze fixation and freeze substitution (1) are a required element in preserving smaller structures that generally escaped our notice in conventional TEM imaging. Modern electron microscopes using higher energy electrons now offer much better resolution by collecting multiple images in a tilting series through comparatively thick sections (150 nm) using the SerialEM program (2). The Protomo software package (3) is used to compute a 3D tomographic reconstruction that offers the same level of detail in any dimension (roughly 2 nm resolution). Annotation and modeling is done using IMOD (4) and/or Amira. This permits us to take a new look at many familiar objects in the anatomy of C. elegans, to identify missing parts of the whole anatomy, and potentially, to detect smaller anatomical defects in a variety of mutant backgrounds. Here we will introduce the tomographic procedure, and share finished 3D models of some typical intracellular organelles at high resolution. Supported by NIH RR 12596 to DHH. 1.Weimer, R.M. (2006) Methods Mol. Biol. 351: 203-221. 2.Mastronarde, D.N. (2005) J. Struct. Biol. 152: 36-51 3.Winkler, H. and Taylor, K.A. (2006) Ultramicroscopy 106: 240-254. 4.Kremer, J.R., Mastronarde, D.N. and McIntosh, J.R. (1996) J. Struct. Biol. 116: 71-76.

Arturo Gutierrez et al. (2003) International Worm Meeting "Posterior Multivulva in Pristionchus pacificus: The ped-9 Case"

Arturo Gutierrez, Ralf J Sommer

[International Worm Meeting, 2003]

The multivulva phenotype in Caenorabditis elegans emerges after mutating specific components of the lin-12/Notch, RAS-MAPK, Wnt or the synmuv pathways. In such mutants, all Pn.p cells of the vulval equivalence group can adopt a 1 or 2 cell fate and develop pseudovulval structures. We are studying vulva development in the Diplogasteridae Pristionchus pacificus to understand the evolution of developmental processes. In a mutagenesis screen in P. pacificus several mutants with a multivulva phenotype were obtained. Interestingly, these mutants are multivulva in the anterior or posterior region, but never in both. One of the complementation groups that was shown to result in a multivulva phenotype is the Hox gene mab-5. In Ppa-mab-5 mutants, P8.p adopts a vulva fate in an M cell dependent manner (Jungblut & Sommer, 1998 and Jungblut et al. 2001). Here we report ped-9, a second posterior locus in P.pacificus that, when mutated, affects the P8.p cell. ped-9 mutant animals show additional phenotypes in the Pn.p cells: P(9-11).p do not undergo apoptosis, contrary to wild type and mab-5 animals, and remain epidermal or differentiated in a gonad-independent way. Epistatic analysis reveals ped-9 as downstream of the Hox gene lin-39 suggesting a function in the regulation of programmed cell death in the posterior region. ped-9 maps on chromosome V, in a region of low recombination. In order to fine-map the gene, recombinant-inbreed lines were developed using the polymorphic strain of Washington (PS 1843) . Currrent work focuses on the AFLP analysis of congenic lines. References: Jungblut B., Sommer J.R. 1998. The Pristionchus pacificus mab-5 gene is involvedin the regulation of the ventral epidermal cell fates. Current Biology, 8: 775-778. Jungblut B., Sommer J.R. 2001. Formation of the egg-laying system in Pristionchus pacificus requires complex interactions between gonadal, mesodermal and epidermal tissues and does not rely on single cell inductions. Development, 128: 3395-404.

Yeh Chan-Hsien et al. (2007) International Worm Meeting "Screen for TLK-1-interacting proteins."

Yi-Chun Wu, Yeh Chan-Hsien, I-Ju Lee

[International Worm Meeting, 2007]

Tousled-like kinases (TLKs) form a conserved serine/threonine kinase family in multi-cellular organisms and have been implicated in transcription, DNA replication and repair and chromosome assembly and segregation. The tlk-1(RNAi) mutants exhibited defects in chromosome condensation and segregation. Previous studies of C. elegans TLK-1 indicates that tlk-1 may act in chromosome assembly by phosphorylating Asf-1 (anti-silencing function protein) and contribute to chromosome segregation as a substrate and activator of the Aurora B kinase AIR-2 (Han et al, 2005). To identify more TLK-1-interacting proteins, we performed a yeast-two hybrid screen. In the screen we isolated DLC-1 (Dynein light chain). DLC-1 is localized to spindle fiber during mitosis and this localization pattern is disrupted in tlk-1(RNAi) embryos. We are currently performing experiments to verify the interaction between DLC-1 and TLK-1 in vivo and investigating if TLK-1 may phosphorylate DLC-1. Reference Han, Z., Riefler, G.M., Saam, J.R., Mango, S.E., and Schumacher, J.M. (2005) Curr. Biol., 15, 894-904.

Costa M et al. (1998) J Cell Biol "A putative catenin-cadherin system mediates morphogenesis of the Caenorhabditis elegans embryo."

Priess JR, Costa M, Raich WB, Hardin JD, Leung BH, Agbunag C

[J Cell Biol, 1998]

During morphogenesis of the Caenorhabditis elegans embryo, hypodermal (or epidermal) cells migrate to enclose the embryo in an epithelium and, subsequently, change shape coordinately to elongate the body (Priess, J.R., and D.I. Hirsh. 1986. Dev. Biol. 117:156- 173; Williams-Masson, E.M., A.N. Malik, and J. Hardin. 1997. Development [Camb.]. 124:2889-2901). We have isolated mutants defective in morphogenesis that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins alpha-catenin, beta-catenin/Armadillo, and classical cadherin, respectively. This putative catenin-cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization.

Rafal Ciosk et al. (2001) International C. elegans Meeting "Maintenance of embryonic stem cell identity in the germline blastomeres of C. elegans"

James Priess, Rafal Ciosk

[International C. elegans Meeting, 2001]

Sexually reproducing organisms arise from germ cells, which have the potential to recreate the whole organism. An important question is how germ cells maintain this potential, or remain totipotent. In early C. elegans embryos, germline blastomeres divide asymmetrically to produce one somatic and one germline daughter. Maternally provided PIE-1 protein is present in the germline blastomeres throughout early development (Mello et al.). Germline blastomeres also contain (but do not respond to) maternally supplied transcription factors that cause differentiation of somatic blastomeres. In pie-1 mutants, germline blastomeres respond to these transcription factors and acquire somatic fates. Thus, PIE-1 is required to prevent germline blastomeres from adopting somatic fates. But what is PIE-1 actually doing? Several lines of evidence suggest that PIE-1 inhibits accumulation of RNA polymerase II-dependent RNAs in the germline blastomeres, and the antibody H14 stains activated RNA polII in somatic cells, but not in germline blastomeres in pie-1 (+) embryos (Seydoux et al.). This phenomenon is referred to as transcriptional inhibition, though the underlying mechanism has not been determined. When PIE-1 is ectopically overexpressed in the somatic blastomeres of the early embryo, it is capable of inhibiting the accumulation of mRNA in those blastomeres (Seydoux et al.). The finding that PIE-1 can cause transcriptional inhibition in all cells of the embryo suggests that PIE-1 targets are present in all cells. We are interested in the possibility of using ectopic PIE-1 in screens to identify additional genes involved in transcriptional repression. We have examined the effect on late embryonic and postembryonic development of a heat shock driven pie-1 transgene (constructed and provided by Geraldine Seydoux). Late stage embryos and adult worms subjected to heat shock express abundant PIE-1 in somatic nuclei, however these same nuclei remain positive for activated RNA polII by H14 immunostaining. Nevertheless, heat shock causes a marked developmental delay that is not observed when larvae are treated with RNAi for pie-1 . To examine the basis for this effect, we are screening for mutants that can suppress the developmental delay and asking whether these have affects on early embryogenesis that are consistent with PIE-1-mediated transcriptional repression. Mello C.C., Schubert C., Draper B., Zhang W., Lobel R., Priess J.R. (1996) Nature , 382(6593):710-2. Seydoux G., Mello C.C., Pettitt J., Wood W.B., Priess J.R., Fire A. (1996) Nature , 382(6593):713-6.

Shelton CA et al. (2000) East Coast Worm Meeting "A C. elegans gene related to S. pombe pom1 is required for microtubule-based processes in the C. elegans embryo"

Creasy CL, Livi GP, Shelton CA

[East Coast Worm Meeting, 2000]

Mutations in the Schizosaccharomyces pombe gene pom1 result in the misplacement of septa, as well as misplacement of microtubule organizing centers during cell division (1). In addition, pom1 mutants are synthetically lethal with mutations affecting either the actin or microtubule cytoskeleton; and the distribution of pom1 protein, Pom1p, within the cell depends on intact microtubules. These observations suggest that Pom1p has a role in positional regulation of the cytoskeleton during division in S. pombe. Pom1p belongs to a protein kinase family with related members in S. cerevisiae, C. elegans, Drosophila, and humans. The functional role of a C. elegans pom1-like gene was examined using RNA interference. 100% lethality was observed among embryos laid by hermaphrodites injected 24 hours prior with dsRNA corresponding to the C. elegans pom1-like gene. A reduced number of severly aneuploid cells were observed in terminal stage embryos. Observations of pronuclear-stage embryos revealed defects in pronuclear migration, spindle orientation, DNA segregation, and cytokinesis. However, actin-based processes such as pseudocleavage formation, cytoplasmic flow, and cleavage furrow formation appeared normal. Therefore, we hypothesize that the C. elegans pom1-like protein is required for microtubule dynamics in the early embryo and, given the similarities between the S. pombe and C. elegans mutant phenotypes, suggest that the yeast and worm genes are functionally conserved. 1. Bahler, J., and J.R. Pringle, Genes and Development, 12(9):1356-1370.

de Vries CJ et al. (1998) European Worm Meeting "A cytosolic dynein heavy chain is required for sensory neuron structure and function."

de Vries CJ, Wicks SR, Plasterk RHA

[European Worm Meeting, 1998]

We have developed two new assays of nematode chemotaxis (see abstract Stephen Wicks). The first gene identified and cloned using these assays was a cytosolic isoform (1B subfamily) of the dynein heavy chain. Complementation studies show that this dynein heavy chain is the CHE-3 protein. Previously, this isoform of the dynein heavy chain was shown to be expressed during cilia outgrowth in sea urchins (Gibbons, 1994), and involved in the organisation of the Golgi-apparatus in non-ciliated human cell lines (Vaisberg, 1996). GFP expression studies indicate that che-3 is expressed exclusively in all ciliated sensory neurons in the worm. The highest levels of che-3 expression are correlated with outgrowth of cilia. To visualise the structure of ciliated endings we used two GFP markers of amphid and phasmid integrity (GPA-13::GFP and GPA-15::GFP, as integrated transgenes, gifts of Gert Jansen). These markers indicate that the amphid and phasmid cilia are malformed in the worm, and that the degree of disorganisation appears to increase as the animal ages. These results suggest that dynein heavy chain has a function in cilia outgrowth in C. elegans, but has no role in the organisation of the Golgi apparatus in the worm. Gibbons, B.H., Asai, D.J., Wen-Jing, Y., Hays, T.S., and Gibbons, I.R. (1994). Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins. Mol. Biol. Cell 5, 57-70. Vaisberg, E.A., Grissom, P.M., and McIntosh, J.R. (1996). Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J. Cell Biol. 133, 831-842.

Wissmann AK et al. (1997) International C. elegans Meeting "Identification of Genes Involved in Small G-Protein Mediated Elongation of the C. Elegans Embryo"

Wissmann AK, Allen FL, Ingles J, Mains PE

[International C. elegans Meeting, 1997]

Elongation of C. elegans embryos appears to be driven by microfilament dependent cell shape changes in the hypodermis (1). Several mutations in let-502 have been identified that lead to arrest during early embryonic elongation (2,3). We have cloned let-502 and found that it encodes a serine/threonine kinase with significant similarity to a group of recently identified kinases activated by the binding of small G-proteins (Rho/Rac) and to human myotonic dystrophy kinase. let-502 mutations can be genetically suppressed by lf mutations in mel-11. We cloned mel-11 and identified its similarity to smooth muscle myosin phosphatase regulatory subunits. We propose a model in which binding of Rho activates LET-502, which in turn inhibits the activity of a smooth muscle-like myosin phosphatase complex via phosphorylation of MEL-11. This inactivation of the phosphatase complex leads to actin-myosin contraction resulting in the cell shape changes necessary for embryonic elongation (3). We have recently obtained evidence that MEL-11 interacts genetically with Rho or a similar G-protein. Double mutants of mel-11 and unc-73, a suspected Rho guanine nucleotide exchange factor, suggest that Rho may have a direct activating effect on MEL-11. We have also conducted a genetic screen for mel-11 suppressors resulting in a number of candidates that may define additional components in this small G-protein mediated pathway. 1 Priess, J.R. & Hirsh, D.L. Developmental Biology 117, 156-173 (1986) 2 Howell, A.M. & Rose, A.M. Genetics 126, 583-592 (1990) 3 Wissmann et al. Genes & Development 11, 409-422 (1997)