Byrne, Alexandra B. et al. (2015) International Worm Meeting "PARGs and PARPs: Novel Regulators of Axon Regeneration."

Miller, III, David M., Byrne, Alexandra B., Hammarlund, Marc, McWhirter, Rebecca D.

[International Worm Meeting, 2015]

Few of the intrinsic mechanisms that regulate axon regeneration after injury are known. To identify genes that regulate axon regeneration, we compared gene expression profiles of FACS-sorted C. elegans GABA motor neurons with high regenerative capacity (conferred by overexpression of DLK-1 MAPKKK) to wild type GABA motor neurons. We detected robust upregulation of both poly(ADP-ribose) glycohydrolases (PARGs), pme-3 and pme-4, in neurons with high regenerative capacity. These data suggest PARG activity might promote axon regeneration. We performed laser axotomy in pme-3 and pme-4 loss-of-function mutants and found that regeneration is impaired. Therefore, PARGs are regeneration-promoting factors.PARGs degrade poly(ADP-ribose), which is synthesized by poly(ADP-ribose) polymerases (PARPs). Thus, the balance between PARG and PARP activity determines cellular levels of poly(ADP-ribose). The PARG-PARP balance regulates multiple processes including DNA damage response, lifespan, and neurodegeneration. Since PARGs counteract PARP function, we hypothesized that loss of PARP activity would have the opposite effect on axon regeneration to loss of PARG activity. We found that loss of function of PARP genes pme-1 and pme-2 increased axon regeneration. Therefore, PARPs inhibit axon regeneration. Together with our PARG findings, these data suggest that levels of poly(ADP-ribose) are a critical determinant of regenerative potential.Next, we investigated whether we could inhibit PARP activity post-injury to promote regeneration of damaged axons. Multiple PARP inhibitors are currently in clinical trials for indications including cancer and stroke. We tested whether PARP inhibitors could enhance axon regeneration. Wild type animals treated with chemical PARP inhibitors after injury showed significantly enhanced axon regeneration compared to controls. Thus, PARP activity regulates the acute response of neurons to axon injury, and PARP inhibition after injury is sufficient to improve regeneration. Together, our findings identify a novel pathway involving control of poly(ADP-ribose) levels that regulates axon regeneration.

Lee, Grace et al. (2021) International Worm Meeting "Microtubule dynamics regulates gap junction trafficking and placement in the motor circuit"

Palumbos, Sierra, Lee, Grace, Strothman, Claire, Miller, III, David M., Zanic, Marija

[International Worm Meeting, 2021]

Gap junctions, or electrical synapses, mediate fast communication between neurons but the mechanisms that direct gap junction assembly between specific neurons or within specific subcellular compartments are poorly understood. Gap junction localization ultimately depends on kinesins, which walk along microtubule (MT) tracks to deliver gap junction components to their cellular destinations. We are investigating gap junction assembly in VA motor neurons in which the UNC-4 transcription factor and its corepressor UNC-37 regulate the neuron specificity and placement of electrical synapses. Gap junction trafficking is disrupted in unc-4 mutants (Palumbos et al, worm meeting 2019). To investigate the mechanism of this effect, we utilized the MT plus-end marker, EBP-2::GFP, to monitor MT dynamics. We determined that the rate of MT polymerization and outgrowth length are reduced in unc-37 mutant VA neurons. VAB-8, an atypical kinesin that binds MTs but lacks motor activity, is negatively regulated by UNC-4/UNC-37. We are now testing the hypothesis that ectopic expression of VAB-8 in unc-4 mutant VA neurons disrupts gap junction trafficking by altering MT dynamics.

Palumbos, Sierra et al. (2021) International Worm Meeting "cAMP controls a trafficking mechanism that directs the neuron specificity and subcellular placement of electrical synapses"

Von Stetina, Steve, Palumbos, Sierra, McWhirter, Rebecca, Miller III, David M, Skelton, Rachel

[International Worm Meeting, 2021]

Despite the functional importance of electrical synapses, the molecular mechanisms that direct the formation of neuron-specific gap junctions remain largely unknown. To address this question, we identified targets of the UNC-4 transcription factor that controls connectivity in the C. elegans motor circuit. UNC-4 functions in VA motor neurons to direct the formation of gap junctions on the VA axon with the interneuron AVA (VA-AVA). Locomotion is disrupted in unc-4 mutants because VAs are miswired with electrical input from the interneuron AVB (VA-AVB) which aberrantly form on the VA soma. Thus, UNC-4 controls both the specificity and subcellular placement of electrical synapses. We determined that UNC-4 blocks expression of two antagonists of cAMP, the phosphodiesterase, PDE-1, and the Go/Gi-coupled GPCR, FRPR-17, to prevent assembly of ectopic VA-AVB gap junctions. This finding suggests that cAMP signaling promotes the formation of functional wild-type VA-AVA gap junctions. We validated this hypothesis by showing that optogenetic elevation of cAMP rescues the Unc-4 movement defect and thus restores VA-AVA circuit function. In addition, forced depletion of cAMP in VAs phenocopies unc-4 mutants. Because gap junction placement is shifted from the VA axon to cell soma in unc-4 mutants, we reasoned that trafficking of gap junction components could be perturbed. Live-cell imaging of the gap junction protein, GFP-UNC-9, confirmed that trafficking into the VA axon is strikingly impaired in unc-4 mutants. Genetic activation of cAMP signaling is sufficient to restore GFP-UNC-9 trafficking in VAs. Thus, we propose that cAMP directs both the specificity and placement of electrical synapses by activating mechanisms that transport gap junction components into the VA axonal compartment. Although studies in cultured cells have implicated cAMP in gap junction assembly, our in vivo experiments now firmly establish that cAMP regulates the biogenesis of electrical synapses in an intact nervous system.

Anthony, Sarah C. et al. (2009) International Worm Meeting "Regulators of synaptic remodeling in C. elegans are revealed by analysis of UNC-55 transcriptional targets."

Miller III, David M., Walthall, Bill, Anthony, Sarah C., Watson, Joseph D.

[International Worm Meeting, 2009]

Neurons form functional circuits by adopting polarized morphologies with separate axonal and dendritic compartments. These domains may be reorganized in response to injury or developmental cues. Though this remodeling feature is evolutionarily conserved, the mechanism is poorly understood. One example of this phenomenon occurs in the GABAergic motor circuit of C. elegans. Dorsal D (DD) motor neurons initially form inhibitory neuromuscular junctions (NMJs) with ventral muscle. At the end of the first larval stage, DDs reverse polarity to synapse with dorsal muscle. This DD polarity switch is coincident with the birth of VD motor neurons, which make NMJs with ventral body muscle. In unc-55 mutants, VD motor neurons are remodeled to mimic the dorsal polarity of DD motor neurons (White et. al., 1978). UNC-55 is a COUP family nuclear hormone receptor normally expressed in VD motor neurons (Zhou and Walthall, 1998). Thus, UNC-55 appears to function as a transcriptional switch to turn off synaptic remodeling genes in VD motor neurons. We propose that UNC-55-regulated targets are likely to fulfill crucial roles in synaptic remodeling. To identify these genes, we employed the mRNA tagging method to compare gene expression profiles of wild-type and unc-55 mutant L2 GABAergic motor neurons. 188 transcripts enriched >2-fold in the unc-55 profile represent candidate synaptic remodeling genes. RNAi of these UNC-55 regulated transcripts revealed >40 genes that partially suppress the Unc-55 remodeling defect (p<0.01), as visualized by rescue of GABAerigc NMJs on the ventral side with the synaptic marker juIs1(punc-25::SNB-1::GFP). Most striking among these candidates is the homeobox transcription factor IRX-1/Iroquois, which suppresses both the loss of ventral NMJs as well as the Unc-55 backward movement defect. Other suppressors of the Unc-55 synaptic remodeling phenotype include specific ion channels, cytoskeletal components, and cell-cell signaling molecules. The wide range of potential functions encoded by these UNC-55 regulated genes is indicative of a complex synaptic remodeling pathway. Ongoing experiments are designed to confirm that these genes lie in the UNC-55 pathway and to investigate their cellular role in the mechanism of synaptic remodeling.

Yuen-Yi Tseng et al. (2007) International Worm Meeting "Caenorhabditis elegans expresses a functional ArsA ATPase."

Vivian Liao, Chan-Wei Yu, Jui-Tung Liu, Yuen-Yi Tseng

[International Worm Meeting, 2007]

As arsenic is the most prevalent environmental toxin, it is imperative for us to understand the mechanisms of metalloid detoxification. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Herein, we describe the identification of a Caenorhabditis elegans homologue (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We have evaluated the responses of wild-type and asna-1 mutant nematodes to various metal ions and found that asna-1 mutant nematodes are more sensitive to As(III) and Sb(III) toxicity than that of wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans'' defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)- ASNA-1 fusion protein was biochemically characterized, and its properties were compared with those of ArsAs. ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 &#177; 0.2 (SEM) fold, while Sb(III) stimulated it by 4.6 &#177; 0.15 (SEM) fold. The results indicate that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria but extend to animals by demonstrating that the asna-1 gene of the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism.

Skelton, Rachel L et al. (2009) International Worm Meeting "unc-4 antagonizes a Wnt signaling pathway upstream of ceh-12/HB9 to specify synaptic choice in the C. elegans motor circuit."

Von Stetina, Stephen, Watkins, Kathie, Skelton, Rachel L, Miller III, David M, Schneider, Judsen

[International Worm Meeting, 2009]

Neural function depends on the creation of synapses between specific neurons. In C. elegans, this important developmental decision is regulated by the UNC-4 homeodomain protein. UNC-4 functions in VA motor neurons to block the adoption of inputs normally reserved for VB sister cells. This wiring defect in unc-4 mutants disables backward locomotion. Thus, we have proposed that UNC-4 preserves normal VA inputs and backward movement by inhibiting VB gene expression. This model was substantiated by our previous finding that ectopic expression of the VB-specific gene, ceh-12 (HB9 homeodomain protein), results in the miswiring of VA motor neurons in the posterior ventral nerve cord. Now we have used cell-specific microarray profiling and independent mutant screens to establish that ectopic expression of CEH-12/HB9 in posterior VA motor neurons depends on a response to a local source of EGL-20/Wnt. Our results show that unc-4 antagonizes a Wnt signaling pathway involving egl-20/Wnt, mom-5/Frz, and mig-1/Frz that is required for ceh-12 expression in VA motor neurons. We propose that UNC-4 represses mom-5/Frz expression, thereby rendering VA motor neurons unresponsive to EGL-20/Wnt. RNAi and genetic experiments have revealed additional Wnt signaling components that interact with the unc-4 pathway, including the Frizzled receptor, lin-17, lin-44/Wnt, dsh-1/Disheveled, pry-1/Axin, and pop-1/TCF-LEF. Further investigation will determine if these canonical Wnt pathway components function upstream of ceh-12 or in a parallel pathway. This work has revealed a sensitive mechanism for exploiting diffusible Wnt ligands for precise patterning of connectivity in the C. elegans ventral nerve cord. The existence of comparable Wnt gradients in the vertebrate spinal cord could reflect similar but as yet unexplored roles for Wnt signaling in vertebrate motor circuit assembly.

Hacker, Mallory L. et al. (2009) International Worm Meeting "The Coenzyme Q Synthesis Gene coq-1 Protects C. elegans GABA Neurons from Calcium-Dependent Programmed Necrosis."

Miller III, David M., Watson, Joseph D., Hacker, Mallory L., Earls, Laurie R.

[International Worm Meeting, 2009]

Coenzyme Q is a component of the mitochondrial electron transport chain and essential for energy metabolism. CoQ deficiency in humans causes cerebellar ataxia and myopathy, indicating that selected tissues are especially sensitive to reduced levels of CoQ. To develop a model for these degenerative diseases in C. elegans, we used RNAi to knock down expression of coq-1, the initial enzyme in the CoQ biosynthetic pathway. RNAi or genetic ablation of coq-1 resulted in a progressive, uncoordinated, or Unc, phenotype and necrotic degeneration of GABA neurons. Both the Unc and degenerative phenotypes emerge during late larval development and progress in adults. Neurons in motor and sensory circuits that utilize other neurotransmitters and body muscle cells did not degenerate upon RNAi depletion of coq-1. The mechanism of GABA neuron cell death depends on the release of intracellular calcium stores and requires the apoptotic genes ced-4 (Apaf-1) and to a lesser extent ced-3 (caspase). Our finding that the mitochondrial fission gene drp-1 (dynamin-related protein) is required for GABA neuron degeneration is consistent with a model in which CED-4 is activated by a signal released from CoQ-depleted mitochondria. Curiously, this pathway is not regulated by the apoptotic genes egl-1 (BH3-only) and ced-9 (Bcl-2) and therefore is likely to include novel components. The progressive and selective degeneration of GABA neurons and the role of mitochondrial dysfunction in CoQ-depleted animals parallels salient features of the human neurodegenerative disorder Huntington''s chorea in which GABA neurons in the brain that control movement are preferentially affected.

Chan-Wei Yu et al. (2007) International Worm Meeting "Characterization and regulation of the C. elegans GCS-1 by arsenite."

Chan-Wei Yu, Vivian Liao

[International Worm Meeting, 2007]

Arsenic is a potent toxin and carcinogen. We previously demonstrated that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress (Yu and Liao, 2005). Herein we investigate the regulation of GCS-1 in response to As(III). Our results showed that As(III) caused a concentration-dependent increase in the GSH levels up to a concentration of 10 <font face=symbol>m</font>M. Real-time RT-PCR analysis shows that gcs-1 mRNA transcript is most abundant in L1, L2 and adult stages. Whereas GCS-1 mRNA at L1 and dauer stages exhibited higher induction ratio after 1 h of As(III) exposure. Moreover, we observed that the increase in GCS-1 mRNA expression level caused by As(III) shows dose-dependence fashion, in parallel with the increase in the GSH level. RNAi feeding assay showed that worms that were fed GCS-1 dsRNA and were grown in the presence of 0.25 mM As(III) were obviously small and development arrest. Transcription of gcs-1 was observed in the pharynx, ASI chemosensory neurons, and intestine of post-embryonic C. elegans and upon exposure to As(III), As(V), or Sb(III), the intestinal expression increases dramatically; whereas Cd(II) induced significant expression in the pharynx. Together, our results indicated that GCS-1 is developmentally regulated and it plays a critical role for the protection of C. elegans against arsenic-induced oxidative stress.

Smith, Cody J. et al. (2009) International Worm Meeting "A combination of morphological landmarks and diffusible cues regulate dendritic arborization in PVD sensory neurons."

Cha, Byeong, Treinin, Millet, Watson, Joseph D., Miller, III, David M., Smith, Cody J., Spencer, Clay W.

[International Worm Meeting, 2009]

Sensory neurons that detect noxious stimuli (nociceptors) display highly branched dendritic arbors adjacent to the skin. The variable complexity of these structures has stymied efforts to identify molecular determinants of dendritic morphogenesis. To simplify this problem, we are using live-cell imaging and genetic methods in C. elegans to study a single type of nociceptive neuron with a stereotypical dendritic architecture. The PVD neuron (L+R) adopts a series of orthogonal branching decisions that envelops the animal in a net-like array of sensory processes directly beneath the hypodermis. Time-lapse imaging with fluorescent markers has revealed that PVD dendrites adopt a 90-degree turn to fasciculate with sub-lateral nerve cords. In contrast, PVD dendrites withdraw upon intracellular contact with an apparent self-avoidance mechanism that insures maximum coverage of the sensory field. These responses suggest that PVD dendritic outgrowth and branching are guided by extracellular signals. We generated a microarray profile of PVD to identify transcripts with potential roles in dendritic morphogenesis. Genetic ablation of the PVD-enriched axon guidance receptors UNC-5 (e152) and UNC-40/DCC (e271) disables the self-avoidance mechanism but does not perturb PVD fasciculation with sub-lateral nerve cords. A similar phenotype was obtained for the axon guidance cue UNC-6/netrin and its downstream effector UNC-34/ena (e315). On the basis of these results, we propose a novel mechanism in which extracellular UNC-6 interacts with UNC-5 and UNC-40 to mediate dendritic self-avoidance. Ongoing experiments are designed to establish the location of UNC-5 and UNC-40 receptors in growing PVD processes and to define the spatial origin of the UNC-6 cue.

Cha, S. et al. (2015) International Worm Meeting "An annotated genome of the root-knot nematode, Meloidogyne chitwoodi."

Cha, S., Bird, D.

[International Worm Meeting, 2015]

Plant-parasitic nematodes are responsible for annual crop losses in excess of USD123 billion worldwide. Most important are the root-knot nematodes (RKN: Meloidogyne spp.), which establish an intimate association with their host. As a genus, Meloidogyne has a host-range that spans the tracheophyta, although individual isolates are more restricted. In cool climates, M. hapla and M. chitwoodi predominate and are a significant problem on potato in Europe and the US. Because of its genetic tractability, we selected M. hapla for sequencing, and an annotated public release (HapPep1) was made in 2008 (Opperman et al., 2008). Since then we have undertaken on-going curation, and the current release (HapPep5) includes ~2xE9 RNA-Seq reads (Guo et al., 2014). We have also sequenced two additional strains of M. hapla (VW8 and LM) but they have not yet been released. Because M. hapla and M. chitwoodi are sympatric, they presumably have similar gene compliments. To test this, we sequenced the M. chitwoodi genome.Genomic DNA was isolated from nematodes collected in a potato field in Washington State, and confirmed by Axel Elling to be M. chitwoodi. Using an Illumina MiSeq II we obtained 20,079,197 short (~300 bp) paired-end reads. The assembly parameters were empirically optimized, and 4,735 contigs assembled; N50 is 82 kbp. The longest is ~758 kbp with coverage of 33 reads per bp. Average coverage genome-wide is 289 reads. At the protein level, CEGMA identified 245 (98.79%) of the core proteins, pointing to near 100% genome coverage. When CEGMA proteins as a query were blasted against the assembled contigs as a database, it was observed that one protein had hits with more than two contigs. Using CEGMA (and other) proteins, as well as M. chitwoodi ESTs, we trained AUGUSTUS for gene prediction. GO categorization was performed using InterProScan. Analysis of these data is in progress, with an emphasis on genes encoding replicas of plant peptide hormones (xenomones). Because of a potential role in speciation of the closely-related parasites, we have, in collaboration with David Lunt (University of Hull), examined the numbers and distribution of transposable elements: not surprising, M. chitwoodi is much more similar to M. hapla than it is to M. incognita.Operman et al., 2008. Proc Nat Acad Sci 105: 14802 -14807.Guo et al. 2014. Worm 3:e29158.