Wang, G. et al. (2017) International Worm Meeting "The mitochondrial signal to non-canonical HIF-1 activation in Caenorhabditis elegans."

Pyonteck, S., Abbott, M., Driscoll, M., Xue, J., Rongo, C., Brook, S., Wang, G.

[International Worm Meeting, 2017]

Transcription factor hypoxia inducible factor-1 (HIF-1) modulates energy metabolism and is well known to be stabilized by hypoxia. However, more and more studies demonstrate that HIF-1 can be stabilized by non-canonical factors, such as oxidative stress, believed to be mediated by hydrogen peroxide (H2O2). Most H2O2 is biologically derived from superoxide (O2-), but O2- is poorly studied as a signaling molecule due to variety of challenges. Superoxide dismutases (SODs) are the only known enzymes to remove O2- in distinct subcellular locations (cytosol, mitochondria or extracellular matrix) of most organisms. Interestingly, deletion of the mitochondrial sod gene in either fly or mouse causes neonatal lethality, but all SODs are dispensable in C. elegans [1], so the worm is a great animal model to study the regulation of life and death by O2-. We found that the HIF-1 signaling pathway is activated in absence of either cytoplasmic or mitochondrial but not extracellular SODs. Moreover, cytoplasmic or mitochondrial O2- showed a synergistic effect on HIF-1 activation. HIF-1 is normally degraded by cytoplasmic prolyl hydroxylases (PHDs) in the presence of O2. a-ketoglutarate (a-KG) also influences activity. Endogenous a-KG is only known to be produced in mitochondria, and it is exported into cytosol via the Malate-Aspartate Shuttle. When we use RNAi to knockdown the two essential Shuttle components, cytoplasmic and mitochondrial malate dehydrogenase, MDH-1 and MDH-2, respectively, we observe activation of the HIF-1 pathway. We speculate that HIF-1 may monitor the change of mitochondrial metabolism via responding to the homeostasis of a-KG, and O2- may stabilize HIF-1 via a similar mechanism, possibly by interacting with positively charged co-enzymes. We examined the pathophysiological role of HIF-1 activation in sod mutants. We found the compartmentalized O2- had totally opposite effects on worm locomotor activities. The thrashing rate is increased in absence of cytoplasmic SODs, but decreased in the absence of mitochondrial SODs. None of these changes are mediated by HIF-1, but mitochondrial O2- can cause more damage without HIF-1. Currently, we are conducting an RNAi screen of the mitochondrial metabolism network genes to map out the mitochondrial signal to non-canonical HIF-1 activation, and testing approaches that reverse O2- mediated HIF-1 stabilization. References: 1. Van Raamsdonk, J.M. and S. Hekimi, Superoxide dismutase is dispensable for normal animal lifespan. Proc Natl Acad Sci U S A, 2012. 109(15): p. 5785-90.

Derek Sieburth et al. (2007) International Worm Meeting "A novel WD repeat protein regulates acetylcholine secretion at the NMJ."

Josh Kaplan, Derek Sieburth

[International Worm Meeting, 2007]

To identify new proteins required for synaptic transmission, we utilized an RNA interference (RNAi) feeding library 1 to knock down predicted C. elegans genes, and screened animals for resistance to the paralytic effects of the acetylcholine esterase inhibitor aldicarb 2. Among the new genes we identified was wdr-23, which encodes a conserved protein that is almost entirely composed of seven WD-40 repeats. We have characterized a putative null, tm1817, generated by the Mitani lab, which deletes 84 amino acids spanning the second and third WD repeats. wdr-23 mutants are lethargic and resistant to aldicarb (the Ric phenotype), but are not resistant to the paralytic affects of the muscle agonist levamisole. wdr-23 is expressed in the nervous system, as well as in body wall muscles3. A wdr-23 cDNA transgene expressed exclusively in the motor neurons is sufficient to fully rescue the locomotion and aldicarb defects of mutant animals. These results suggest that wdr-23 acts in motor neurons to promote acetylcholine release at the neuromuscular junction (NMJ). Several results suggest that WDR-23 plays a central role in regulating acetylcholine secretion at the NMJ. First, knockdown of wdr-23 by RNAi blocks acetylcholine secretion under a variety of different conditions that normally enhance neurotransmission4. Second, WDR-23 fused to GFP becomes highly punctate in axons of mutant animals that are defective in exocytosis, suggesting that WDR-23 dynamically associates with presynaptic terminals. Finally, we found a significant accumulation of the synaptic vesicle protein synaptobrevin tagged with GFP (GFP::SNB-1) in wdr-23 mutant synapses compared to wild type. GFP::SNB-1 accumulation was similar to that observed in known exocytic mutants unc-13 and unc-18. Together these results suggest that WDR-23 is a novel, conserved, regulator of neurotransmiter secretion that is required for synaptic vesicle exocytosis. 1. Fraser, AG et al. Nature 408, 325-30 (2000). 2.Miller, KG et al. Proc Natl Acad Sci U S A 93, 12593-8 (1996). 3. www.wormbase.org 4.Sieburth, D et al. Nature 463, 510-517 (2005).

Zamberlan, D. C. et al. (2013) International Worm Meeting "The Rosmarinus officinalis extract protects against oxidative stress and increase C. elegans lifespan."

Zamberlan, D. C., Soares, F. A. A., Amaral, G. P., Puntel, R. L., Stefanello, S. T.

[International Worm Meeting, 2013]

Oxidative stress has been associated to many genetic and acquired disorders and ageing processes. The Rosmarinus officinalis L. is known for it uses in food recipes, but it has also gaining interest by its pharmacological properties. The ethanolic extract of R. officinalis L. (eeRo) exhibited significant antioxidant, vasodilator and antiinflammatory properties. The aim of this study was to analysis the possible protective propriety of eeRo in the nematodeo C. elegans. The Rosmarinus officinalis leaves were subjected to an alcoholic extraction. HPLC fingerprinting of eeRo revealed the presence of the chlorogenic acid (12.2 mg/g), caffeic acid (7.63 mg/g), rutin (3.07mg/g), rosmarinic acid (38.5 mg/g), quercetin (5.10mg/g), kaempferol (2.53 mg/kg), and carnosic acid (26.4 mg/g). The worms were treated with the extract (1, 2.5 or 5 mg/ml) added to de plate and kept at 20oC. The wildtype strain N2 survival was analyzed under oxidative stress and heat stress. The transgenic strain TK22, wich is oxygen sensitive and has a shorter lifespan compared to the wild type was also used. The effects of eeRo on TK22 lifespan and reactive oxygen species (ROS) production were analysed. In lifespan assay the worms were scored as dead or alive by touch evoked movement every two days until the last worm dies. The ROS production was measured by CM-H2DCFDA. The extract in all concentration was able to decrease the ROS production in N2 and TK22 worms with 2.5 mg/ml being the most efficient. The treatment with eeRo 2.5 mg/ml also protected the worms against oxidative and heat stress increasing the worm survival by 37% and 27.8% respectively. Moreover, the worms treated with eeRo 2.5 and 5 mg/ml showed an increase in the mean and total lifespan of TK22 mutant strain when compared with untreated controls. The results demonstrate that the eeRo has antioxidant activity in vivo, inducing decreased of the ROS, increase of stress surviving, besides provides thermotolerance. However, the exact mechanism by which it exerts its protection remains unclear.

Y Zheng et al. (1999) International C. elegans Meeting "glr-3 Encodes a non-NMDA Type Glutamate Receptor that is Widely Expressed in the Nervous System of C. elegans"

AV Maricq, Y Zheng

[International C. elegans Meeting, 1999]

We have shown that a defect in a glutamate receptor subunit, glr-1 , differentially affects the transmission of sensory input from the polymodal sensory neuron ASH (see also Hart et al, 1995). Mutants are defective in the backing response to tactile stimuli, but have normal responses to osmotic stimuli. One possible explanation for this interesting phenotype is that other glutamate receptors are required for the proper transmission of sensory input from ASH. One non-NMDA subtype of glutamate receptor that we have identified is glr-3 . We are most interested in glutamate receptors found in the command interneurons. Based on transgenic strains that express fusions of glr-3 with GFP, we have shown that glr-3 is expressed in the command interneurons as well as many other neurons, including neurons in the ventral cord. We have isolated a full-length cDNA for glr-3 that encodes a 932 a.a. receptor with 4 predicted transmembrane (TM) domains. GLR-3 has many of the topological features of glutamate receptors and shows strong conservation in the predicted TM domains. It has some unusual features however, including a KG sequence in the highly conserved residues, (Q/R Q), that are found in TM II of most non-NMDA receptors. The GFP expression data obtained from transgenic strains shows that glr-3 is expressed in some of the same command interneurons that express glr-1 , glr-2 , glr-4 , nmr-1 , and nmr-2 . To evaluate the contribution of this receptor to behavior we have intiated experiments designed to generate a null mutation. We have identified two strains that contain a Tc1 transposable element in the glr-3 gene: one inserted in an intron, the other in an exon. We are now in the process of generating deletion mutations by imprecise excision of these Tc1 elements. We will examine the behavior of these mutants as well as the behavior of strains with combinations of receptor mutations. How does glr-3 contribute to the function of these neurons? To begin to address this question we will determine the subcellular location of this receptor subunit. In particular, we are interested in determining whether glr-3 co-localizes with any of the other receptor subunits. This localization data will help predict which receptor subunits form functional heteromeric receptors.