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.

Bond, Michelle R et al. (2011) International Worm Meeting "O-GlcNAc cycling is a regulator of innate immunity in Caenorhabditis elegans."

Krause, Michael W, Hanover, John A, Bond, Michelle R

[International Worm Meeting, 2011]

Playing critical roles in biological process including signaling and gene transcription, the O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification of serine and threonine residues is dynamic. Modifying and/or regulating over 600 proteins, O-GlcNAc has been implicated in a variety of human conditions including Diabetes mellitus, cancer, and neurodegenerative diseases. O-GlcNAc cycling is governed by two proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that add and remove the modification, respectively. OGT interacts with evolutionarily conserved proteins that are key for the innate immune response: it interacts with components in the beta-catenin, p38 MAPK, and insulin signaling pathways. Given its role as a key signaling molecule and the pathways with which OGT interacts, we hypothesize that O-GlcNAc cycling plays a role in "fine-tuning" the innate immune response. Bacterial infections, especially those that are drug resistant, are of growing health concern in the USA. Although significant progress has been made in understanding organisms' responses to bacterial infections, the complexity of the mammalian system leaves in-depth characterization of immune pathways at the whole-organism level wanting. The bacteriovore C. elegans is an attractive candidate for studying the innate immune response: it is susceptible to many human pathogens, the animal's transparency allows real-time observation of bacterial accumulation and its short life span permits characterization of susceptibility by scoring longevity. Importantly, although the removal of OGT and OGA yields lethality in most organisms, null mutants of OGT and OGA are viable and fertile in C. elegans. The innate immune response was examined for wild type (WT), ogt-1 mutant, or oga-1 mutant worms to heat-killed S. aureus (HKSA). While the WT worms had similar longevity profiles regardless of the bacterial strain they were fed, ogt-1 and oga-1 mutant worms had an increased lifespan on HKSA in comparison to WT. These data suggest that defects in O-GlcNAc cycling may influence the animals' sensitivity to bacteria by changing the proteins modified by O-GlcNAc. In order to understand the implications of these data, we study the C. elegans innate immune response to live S. aureus and profile the proteins modified by O-GlcNAc in each case by immunoblot and mass spectrometry. The susceptibility of both O-GlcNAc cycling mutants and other innate immunity mutants is currently under investigation.

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.

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)

Peng Wang et al. (2009) International Worm Meeting "O-GlcNAc modification and proteotoxicity in Caenorhabditis elegans."

Peng Wang, Brooke D. Lazarus, Michael W. Krause, John A. Hanover, Michele E. Forsythe, Dona C. Love

[International Worm Meeting, 2009]

O-linked N-acetylglucosamine (O-GlcNAc) addition is a type of 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. Disruption of the O-GlcNAc cycling is involved in several human diseases, including diabetes mellitus and neurodegenerative diseases. One common signature of neurodegenerative diseases is the formation of protein aggregates, with toxic effects that could lead to neuronal degeneration and death. Human proteins that form protein aggregates in different neurodegenerative diseases have been expressed in the neurons or muscles of C. elegans to construct transgenic models. These human proteins include the following: different isoforms of Tau proteins (Alzheimers disease), beta-Amyloid (Alzheimers disease), Huntingtin (N-terminal)-polyglutamine fusion protein (Huntingtons disease), and yellow fluorescent protein (YFP)-polyglutamine fusion protein (PolyQ diseases). To investigate the effects of O-GlcNAc modification on proteotoxicity in a whole model organism, we crossed those Caenorhabditis elegans transgenic models into ogt-1 or oga-1 null mutant. The phenotypes of the transgenic animals were scored in wild type, ogt-1 mutant, or oga-1 mutant background. We found that the ogt-1 mutation alleviated and the oga-1 mutation worsened the phenotypes that are caused by the toxicity of those human proteins. In addition, in a Huntingtons disease model, we observed the same rescued phenotypes in ogt-1; oga-1 double mutant as in ogt-1 single mutant, indicating that alterations of the phenotypes depend on O-GlcNAc modification. The level of transcript of the transgenes, measured by SYBR green real-time PCR, did not correlate with the alteration of phenotypes that we observed in different genetic backgrounds. Thus, regulation of some protein quality control systems by O-GlcNAc modification might be the underlying mechanism. There are two main protein degradation pathways: proteasome and autophagy. Our working hypothesis is that both degradation pathways might be up-regulated in ogt-1 mutant and down-regulated in oga-1 mutant.

Wang P et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Loss Of O-GlcNAc Is Neuroprotective In Caenorhabditis Elegans Models Of Human Neurodegenerative Diseases"

Hanover J A, Lazarus B D, Krause M K, Forsythe M E, Wang P, Love D C

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

The O-linked N-acetylglucosamine (O-GlcNAc) modification of serine or threonine hydroxyl group is a dynamic, post-translational process mediated by two evolutionally conserved enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add or remove O-GlcNAc, respectively. This process modifies and regulates more than 600 proteins. Deregulation of this process is involved in various human diseases including diabetes mellitus, cancer, and neurodegenerative diseases. One common signature of neurodegenerative diseases is the formation of protein aggregates, which lead to neuronal degeneration and death. Ectopically expressing aggregate-prone human proteins in C. elegans neurons or muscles provides a whole and simple model organism for studying these complicated diseases. The transparency of the worm allows real-time observation of protein aggregates in live animals. And the short life span of C. elegans makes it more feasible to investigate the effects of aging on neurodegeration. Null mutants of OGT or OGA in C. elegans are viable and fertile, compared to lethal in mammals, making it a good model system to investigate the effects of deregulation of O-GlcNAc modification in C. elegans neurodegenerative models. Proteotoxicity in different C. elegans models was compared in wild type, ogt-1 mutant, or oga-1 mutant background. We found that the ogt-1 mutation alleviated and the oga-1 mutation enhanced the toxicity of aggregate-prone proteins such as FTDP-17 Tau, beta amyloid, and polyglutamine (polyQ). Furthermore, the enhanced proteotoxicity of polyQ found in oga-1 mutants was reversed by ogt-1 mutation, indicating that absence of O-GlcNAc modification leads to reduction of the aggregate-associated proteotoxicity. Our data also showed elevated proteasome activity in ogt-1 mutants, suggesting a faster turnover of the protein aggregates accounts for the phenotypic alleviation observed in this background. The regulation of O-GlcNAc modification of another protein quality control pathway, autophagy, is currently under investigation. These results indicate that OGT and OGA counteract each other in regulating proteotoxicity in multiple neurodegenerative disease models, and regulation of protein quality control pathways by O-GlcNAc modification could be the underlying mechanism.