Wang, Peng et al. (2011) International Worm Meeting "Caenorhabditis elegans O-GlcNAc cycling mutants alter the proteotoxicity of models of human neurodegenerative disorders."

Forsythe, Michele, Wang, Peng, Lazarus, Brooke, Hanover, John, Love, Dona, Krause, Michael

[International Worm Meeting, 2011]

O-linked N-acetylglucosamine (O-GlcNAc) addition is an important post-translational modification that occurs on hundreds of proteins, including nuclear pore proteins, transcription factors, proteasome components and neuronal proteins. O-GlcNAc can be added onto and removed from serine or threonine residue by two evolutionally conserved enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. O-GlcNAcylation is abundant in the brain and it has been linked to human neurodegenerative disease. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized C. elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the proteotoxic phenotype in transgenic models of tauopathy, b-amyloid peptide and polyglutamine expansion. Intriguingly, loss-of-function of OGT alleviated, while loss of OGA enhanced these proteotoxicity phenotype. Consistent with these observations, the O-GlcNAc cycling mutants exhibit altered stress responses and changes in the protein degradation machinery. These findings suggest that modulators of O-GlcNAc cycling may prove useful for anti-neurodegenerative disease therapies.

Lee, J. et al. (2009) International Worm Meeting "Proteomic analysis of O-GlcNAcylation during dauer formation in C. elegans."

Paik, Y., Kim, K., Lee, J.

[International Worm Meeting, 2009]

Modification of proteins at serine or threonine residues with N acetylglucosamine (O-GlcNAcylation) plays a regulatory role in many model organisms. Here, we investigated the mechanism by which O-GlcNAcylation regulates entry into the stress-induced dormant state dauer in Caenorhabditis elegans. We confirmed that ogt-1 (O-GlcNAc transferase) mutants exhibited a dauer-defective phenotype whereas oga-1 (O-GlcNAcase, catalyzes O-GlcNAc removal) mutants exhibited a dauer-prone phenotype when treated with daumone. Consistent with these findings, treatment with low levels of daumone and the O-GlcNAcase inhibitor PUGNAc enhanced the frequency of dauer entry. Treatment of daf-2 with PUGNAc increased the frequency of dauer entry, providing additional evidence that O-GlcNAcylation promotes dauer formation. Alterations to the proteome as a result of induction of O-GlcNAcylation were analyzed by two-dimensional electrophoresis (2DE) using lysates of N2 and oga-1 worms. Seven differentially expressed protein spots were further analyzed using LC-MS/MS. The identities of these proteins suggest that O-GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. To identify potential target proteins that are O-GlcNAcylated during dauer formation, we specifically labeled O-GlcNAc with fluorescent dye using O-GlcNAc labeling system containing TAMRA fluorescence dye targeting O-GlcNAcylated proteins. The fluorescently labeled samples were resolved by 2DE and 20 protein candidates were selected. Our data suggest that O-GlcNAcylation may regulate a shift in some proteins including cytoskeletal and protein turnover during dauer formation. Thus, these data may be valuable in identifying the mechanism by which high levels of O-GlcNAcylation enhance dauer formation. (This study was supported by a grant from the Korea Health 21 R&D project, Ministry of Health and Welfare of Republic of Korea [A030003 to YKP].).

Mahaffey, M. et al. (2017) International Worm Meeting "O-GlcNAc cycling and mitochondrial function."

Mahaffey, M., Austin, B., Berninsone, P., Sacoman, J.

[International Worm Meeting, 2017]

O-linked- beta -N-acetylglucosamine (O-GlcNAc) modified proteins are critical in myriad cellular processes and functions. Abnormal O-GlcNAcylation is associated with a spectrum of diseases, including Alzheimer's disease, cancer, cardiovascular disease, and diabetes. Mitochondrial protein O-GlcNAcylation is emerging as a key regulator of cellular energetic metabolism, redox signaling and cell survival pathways, but the mechanisms involved are largely unknown. O-GlcNAc transferase (OGT) is the enzyme responsible for the addition of O-GlcNAc to target proteins while O-GlcNAcase (OGA) catalyzes the removal of the modification from target proteins. OGT and OGA are encoded by single genes in C. elegans (ogt-1 and oga-1, respectively). OGT and OGA modulate lifespan, stress susceptibility and oxidative stress resistance. We hypothesize that O-GlcNAc cycling mediated by OGT and OGA plays a role in regulating mitochondrial metabolism and morphology. To that end, we measured oxygen consumption rate (OCR), a proxy for mitochondrial oxidative phosphorylation function, in N2, ogt-1 and oga-1 null mutants. OCR was measured using the Seahorse Bioscience XFe 24 Extracellular Flux Analyzer and normalized by number of worms for each individual well. Our results suggest that altered O-GlcNAc cycling reduces oxygen consumption by negatively impacting mitochondrial oxidative metabolism. In addition, O-GlcNAc cycling mutants have altered sensitivity to rotenone, an inhibitor of NADH ubiquitone reductase (Complex I). These results suggest that modulation of mitochondrial function by O-GlcNAc cycling may underlie some of the phenotypes of O-GlcNAc cycling mutants.

Scott Everet Baird et al. (2004) East Coast Worm Meeting "Association of Oscheius species with millipedes in the Wright State University Woods."

Scott Everet Baird, Christine M Spice

[East Coast Worm Meeting, 2004]

Rhabditid nematodes commonly form phoretic or necromenic associations with other soil invertebrates. These associations typically are as dauers although although associations of other larval stages also have been reported. Two hermaphroditic species of Oscheius , O. myriophila and an isolate tenatively identified as O. necromena , have been reported as necromenic associates of millipedes. Both of these species have been found in association with the millipede Pseudopolydesmus serratus in the Biology Preserve on the campus of Wright State University. A third as yet unnamed hermaphroditic species of Oscheius was found in association with a second millipede, Oxidus gracilis . O. myriophila , O. necromena , and O. sp. were found at the same collection site, i.e. they were sympatric. O. myriophila never was found in association with Ox. gracilis despite this millipede being its type host. When first observed, all collected nematodes were L4s, consistant with a previous report of the association of O. myriophila L4s with millipedes. Relationships among these species were investigated through sequence comparisons of 18S rDNA. O. myriophila , O. necromena , and O. sp. clustered together as a monophyletic clade within the insectivora -group of Oscheius . Thus, association with millipedes and their hermaphroditic mode of reproduction may be ancestral characters of this group of species.

Rahman, Mohammad M. et al. (2009) International Worm Meeting "Elevated O-GlcNAc modification can extend Caenorhabditis elegans lifespan."

Rahman, Mohammad M., Wells, Lance, Karim, Enas, Stuchlik, Olga, Kipreos, Edward

[International Worm Meeting, 2009]

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification is an abundant nucleo-cytoplasmic post-translational glycosylation of proteins associated with age-related diseases like Alzheimer''s, Parkinson''s, and type II diabetes. However a link between O-GlcNAc modification of proteins and organismal aging has not been demonstrated. This work uses the nematode C. elegans to establish a link between nutrient availability, O-GlcNAc cycling, and longevity. We found that O-GlcNAc modification of protein(s) is critical for normal lifespan in adult animals, while unchecked O-GlcNAc modification of protein(s) increases adult lifespan in C. elegans. We demonstrate that the adult lifespan extension arising from an elevated level of O-GlcNAc modification is dependent on the DAF-16/FoxO transcription factor. DAF-16 is a key factor in insulin-like signal transduction, which regulates reproductive development, lifespan, and dauer formation in response to nutrient availability. Our current data indicates that O-GlcNAc cycling influences a subset of insulin-like signaling-mediated functions that regulate adult lifespan, without affecting other downstream aspects of the insulin-like signaling pathway.

Taub, D.G. et al. (2019) International Worm Meeting "O-GlcNAc signaling enhances neuronal regeneration through modulation of cellular metabolism."

Awal, M.R., Taub, D.G., Gabel, C.V.

[International Worm Meeting, 2019]

The lack of neuronal regeneration after damage to the central nervous system remains a critical deficiency in modern medicine. While regrowth of a severed axon is inherently energetically demanding, the mechanisms of metabolic regulation in a damaged neuron remain largely unknown. This represents a critical gap in our understanding of neuronal repair that is fundamental to therapeutic strategies. We have recently discovered that alterations in O-GlcNAc signaling can shift cellular metabolism to dramatically potentiate the regenerative capacity of a damaged neuron in vivo. O-linked ?- N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serines/threonines that functions as a sensor of cellular nutrients. Performing in vivo laser axotomies in C.elegans, we find that neuronal regeneration is substantially increased by disruptions of either the O-GlcNAc Transferase or the O-GlcNAcase that decrease and increase O-GlcNAc levels, respectively. A lack of O-GlcNAc acts through the AKT-1 branch in the insulin-signaling pathway to utilize glycolysis. In contrast, increased O-GlcNAc levels activate an opposing branch of the insulin-signaling pathway whereby SGK-1 modulates the FOXO transcription factor DAF-16 to influence mitochondrial function. Exploiting the effects of O-GlcNAc signaling on metabolism and regeneration, we are determining the metabolic response within a damaged neuron, how cellular metabolism acts as limiting factor in neuronal regeneration and how it might be altered for neuro-therapeutic benefits.

Stefinko, M. et al. (2017) International Worm Meeting "Identifying new components of the EGL-15 fibroblast growth factor receptor signaling pathway."

Lo, T.-W., Voisine, C., Stefinko, M., Payan Parra, O., Eichelberger, E., Puccini de Castro, V., Stern, M.J.

[International Worm Meeting, 2017]

The study of the EGL-15 fibroblast growth factor receptor (FGFR) in C. elegans has long served as a paradigm for understanding principles of receptor tyrosine kinase (RTK) signaling. Defects in the processes mediated by EGL-15 result in striking phenotypes that provide powerful genetic tools that have been used to discover many components that mediate RTK signaling. One example is the regulation of fluid homeostasis - hyperactivation of EGL-15 results in a Clr (Clear) phenotype characterized by excessive accumulation of clear fluid within the worm's body. Hyperactivation of EGL-15 signaling is typically accomplished by a mutation in the gene clr-1, which encodes a receptor tyrosine phosphatase that negatively regulates the EGL-15 signaling pathway. The isolation of suppressors of the Clr phenotype, termed Suppressor of Clr (soc) mutants, has led to the identification of many of the core components of EGL-15 signaling. For example, the Grb2/SEM-5 adaptor protein that links RTK activation to the activation of the RAS GTP-binding protein was identified in the original set of soc mutations. Although SEM-5 is required for the regulation of fluid homeostasis via EGL-15, a key signaling component that links activated EGL-15 to SEM-5 has yet to be identified. While activated EGL-15 can recruit SEM-5 via phosphorylated tyrosines in its carboxy-terminal domain (CTD), this mechanism cannot explain the SEM-5 requirement for fluid homeostasis, since an egl-15 mutation, n1457, that truncates the CTD and eliminates these binding sites does not confer a Soc phenotype. To identify these missing components, we conducted a modified, "enhancer" soc screen in an egl-15(n1457 DCTD) background and identified 33 new soc mutations. Five of these are alleles of known, major components of the EGL-15 signaling pathway: four alleles of egl-15 itself, and one allele of soc-1. Preliminary characterization of eleven additional new soc enhancer mutations revealed that two are autosomal; the other nine are X-linked and define at least two new soc genes. We have identified multiple alleles of one of these genes; each of these alleles has a weak Soc phenotype on its own, but a strong Soc phenotype in the enhancer background. The enhancing effect suggests that this new gene likely cooperates with other components of the EGL-15 signaling pathway. Whole-genome sequencing will be used to identify the molecular identity of these new signaling genes. The continued characterization of these soc mutations will help further our understanding of the molecular mechanisms by which RTKs promote specific biological responses to intercellular signals.

Carta LR et al. (1995) International C. elegans Meeting "NEW HERMAPHRODITIC SPECIES OF OSCHEIUS (ANDRASSY, 1976) (RHABDITIDAE, NEMATODA)"

Thomas WK, Carta LR, Sternberg PW

[International C. elegans Meeting, 1995]

Morphological, life cycle and molecular characteristics are used to describe seven hermaphroditic species of Oscheius (Andrassy, 1976) including O. brevesophaga, n. sp. O. mesoesophaga n. sp., O. fijiensis n. sp., O. spiculunca n. sp. O. longirecta n. sp., O. subvulva n. sp., and O. longimascula n. sp. These were compared with other living isolates of the group provided through David Fitch. Starvation induced males were mated among the isolates to define distinct biological species. These species were sequenced over a 310 nucleotide bp region of the nuclear 28S rRNA. Vulval anatomy was observed in the early L4 stage. Life cycle was determined from egg to egg at 20 C. to within half a day. The only non-fertile hybrids produced were those of O. mesoesophaga hermaphrodites mated with O. tipulae males. With just 1 base pair difference between each sp. in the series, O. brevesophaga, mesoesophaga and tipulae are best distinguished by esophageal length. By contrast, three interbreeding isolate groups of O. brevesophaga are defined by 1 or 2 bp changes. Thus, at least 2 bp changes may be necessary to distinguish a biological species in this group, while no change may be sufficient (e.g. as in Rhabditis anomala and a new hermaphroditic sibling species, Rhabditis heranamaloides). This can help define species where males are nearly impossible to find or mate. Vulval anatomy revealed that 2o lineages generated 4 cells and 3o generated 4 cells (4:4) in the three non- Dolichura groups, and 4:2 cells in three of the four species of the Dolichura group examined. (Caenorhabditis has a 7:2 pattern.) A significant number of O. subvulva individuals showed an interesting asymmetry with 4 cells resulting from the 3o P4.p lineage, while the 3o P8.p lineage gave only two cells. Four major groups are defined by larger base pair difference gaps between than within groups. These correspond to somewhat distinct groups morphologically and developmentally as follows: Tipulae group: Spicules <30 micron with tips not visibly swollen, Life cycle < 5 days at 20 C, 2o:3o = 4:4. (O. brevesophaga, mesoesophaga, tipulae) Insectivora group: Spicule tips hooked or acute, Spicule > 30 micron, Bursa usually pseudopeloderan, >= 4 lateral lines, life cycle < 5 days at 20 C, 2o3o = 4:4. (O. longirecta, spiculunca) Fijiensis group: Spicule tips swollen, life cycle < 5 days at 20o C, Medial vulva, 2o:3o = 4:4. (O. fijiensis) Dolichura group: Spicules with swollen tips, Life cycle > 5 days at 20o C, Vulva usually > 50%, 2o:3o usually = 4:2. (O. dolichura, dolichuroides, subvulva, longimascula)

Eustice, Moriah et al. (2015) International Worm Meeting "O-GlcNAc cycling plays a crucial role in germline plasticity and acts in coordination with fatty acid beta-oxidation to regulate ARD entry in C. elegans."

Hanover, John, Eustice, Moriah

[International Worm Meeting, 2015]

Adult reproductive diapause (ARD), or the oogenic germline starvation response, is triggered when C. elegans are starved at the mid-L4 stage of development. ARD is characterized by extended lifespan and reproductive potential as well as germline plasticity, wherein the germline shrinks back to approximately 35 germ cell nuclei per gonad arm during starvation but regenerates following refeeding. Previous studies found that the fatty acid beta-oxidation protein NHR-49 plays a key role in the establishment of ARD. However, questions remain about the way in which changes in metabolism are sensed and trigger the plasticity observed with adult diapause.To address these questions, we aimed to examine the complex relationships between ARD and metabolism, specifically focusing on the modification O-N-acetyl-glucosamine (O-GlcNAc). O-GlcNAc is a dynamic monosaccharide post-translational modification that integrates signals from several metabolic pathways, including glucose and fat metabolism. Key enzymes in the cycling of O-GlcNAc are O-GlcNAc transferase (OGT), which is responsible for the addition of O-GlcNAc, and O-GlcNAcase (OGA), which removes the modification. Due to the fact that the substrate of OGT, UDP-GlcNAc, is produced as the final step of the hexosamine biosynthetic pathway, the regulation of O-GlcNAc cycling is uniquely situated to link metabolic signaling with growth, development, and longevity. Using mutants of OGT and OGA we discovered that O-GlcNAc cycling plays a prominent role in both ARD entry and exit. Furhter, we found that the role of O-GlcNAc in the establishment of ARD is genetically linked to NHR-49 function, such that impairment of O-GlcNAc cycling suppresses the NHR-49 entry defect.These findings suggest that perturbation of nutrient-sensitive O-GlcNAc is crucial to the developmental dynamics observed in ARD and acts in concert with NHR-49 to regulate entry into reproductive diapause. Understanding ARD dynamics is important as it provides a unique model in which to explore the relationship between nutrient status and the regulation of reproduction and aging. .

Ceballos, Ainhoa et al. (2015) International Worm Meeting "Transcriptional regulation of metabolic genes by promoter-localized O-GlcNAc."

Cecere, Germano, Ceballos, Ainhoa, Grishok, Alla

[International Worm Meeting, 2015]

The O-Linked-N-acetylglucosamine (O-GlcNAc) modification of proteins is one of the most abundant in eukaryotes. It is very sensitive to glucose levels and environmental conditions and therefore has the potential to lead environmentally-induced epigenetic changes. In C. elegans, prominent peaks of O-GlcNAc were identified close to transcription start sites (TSS) of many metabolic genes by ChIP/chip, although the identitiy of the O-GlcNAc modified protein is unknown (Love et al.,2010). We described a negative regulation of transcription of growth-related and metabolic genes by the conserved chromatin-binding factor ZFP-1 and its interacting partner the histone methyltransferase DOT-1.1 (Mansisidor et al.,2011; Cecere et al., 2013). Intriguingly, ZFP-1 and DOT-1.1 ChIP-chip peaks significantly overlap with O-GlcNAc ChIP-chip peaks at TSS of active genes. Although our extensive analyses determined that ZFP-1 and DOT1.1 do not carry this modification, we have found that the level of O-GlcNAc on chromatin is elevated in zfp-1 mutant worms, which correlates with the increased transcription of ZFP-1 target genes. On the other hand, we have determined that promoter-localized O-GlcNAc stimulates transcription of the same genes. These results indicate that ZFP-1 and O-GlcNAc have opposite roles in regulating transcription of metabolic genes. We are currently focusing on a candidate protein modified by O-GlcNAc and will present our progress in understanding its interplay with the ZFP-1/DOT-1.1 complex at the glucose-responsive promoters.