Gandhi, Jay et al. (2021) MicroPubl Biol

Fernandez, Anita G., Gandhi, Jay, Crosio, Giulia

[MicroPubl Biol, 2021]

Most cellular processes require the coordinate activities of multiple gene products. These collaborations vary with different contexts. mel-28 encodes a conserved multi-functional nucleoporin required for nuclear envelope integrity, the post-mitotic rebuilding of the nuclear pore, and the proper segregation of chromosomes during mitosis (Galy et al. 2006; Fernandez and Piano 2006). Loss of mel-28 results in strict maternal-effect embryonic lethality. Thus homozygous mutant embryos produced from heterozygous mothers show typical wild-type development until they become adults and produce normal-sized broods of inviable embryos. This suggests that mel-28 is required for embryonic development but is dispensable for post-embryonic development and adult processes such as fertility. To identify genes that function collaboratively with mel-28, we did an RNAi screen to find genes that cause fertility defects in mel-28 mutant animals but not wild-type animals (Fernandez et al. 2014). Among the genes identified were those encoding components of the minus-end-directed microtubule motor dynein.

Giorgi, Victoria et al. (2021) International Worm Meeting "C. elegans as a model for studying Cannabis sativa extracts"

Garcia, Carlos, Carrera, Ines, Fernandez, Santiago, Castro, Rosina, Giorgi, Victoria, Vila, Agustina, Pazos, Mariana

[International Worm Meeting, 2021]

The Cannabis genus includes a variety of plants that are a valuable source of secondary bioactive metabolites, including flavonoids, terpenes, and cannabinoids. These metabolites are responsible for a wide variety of biological activities such as anti-inflammatory, anti-oxidant, psychotropic and others. The long term objective of this project is to develop C. elegans as a model for studying the bioactive potential of different extract types from cannabis plants. Previous studies showed that aqueous extracts from Cannabis sativa leaves exhibit nematicidal activity on plant parasitic nematodes, and suggested that phytocannabinoids present in the extract are responsible for this effect. However, little is known about the nematicidal activity from extracts of other parts of cannabis plants, including female flowers where phytocannabinoids accumulate in highest amounts. C.elegans has proven to be an excellent model for the discovery of new anthelmintic drugs and its mechanism of action. In this sense extracts from flowers of two Cannabis plant varieties which differ in its ratio of tetrahydrocannabinolic acid (THCA)/ cannabidiolic acid (CBDA) content were prepared and its activity on C. elegans motility assessed. The phytocannabinoids profile of the extracts was studied and the main phytocannabinoids were identified and quantified by HPLC-DAD. Automated motility assays were conducted with N2 worms at 10 microM and 100 microM in the main cannabinoid (CBDA or THCA). Failure to see a motility effect in this background prompted us to use mutants with a compromised cuticle. C.elegans has a robust cuticle that functions as a physical barrier against chemicals which may represents a problem for compounds testing, such as acidic phytocannabinoids. In this sense, motility assays were performed using mutants in the bus-8 gene, which encodes a predicted glycosyltransferase required for cuticle integrity. Cannabis flower extracts caused severe reduction in motility in bus-8 mutants at 50 microM and 100 microM in the main cannabinoid. We are currently analyzing purified compounds to dissect the complex effect of these extracts and using mutant analysis to understand its mechanism of action.

Inberg S et al. (2019) Annu Rev Neurosci "Lessons from Worm Dendritic Patterning."

Meledin A, Kravtsov V, Oren-Suissa M, Inberg S, Iosilevskii Y, Podbilewicz B

[Annu Rev Neurosci, 2019]

The structural and functional properties of neurons have intrigued scientists since the pioneering work of Santiago Ramon y Cajal. Since then, emerging cutting-edge technologies, including light and electron microscopy, electrophysiology, biochemistry, optogenetics, and molecular biology, have dramatically increased our understanding of dendritic properties. This advancement was also facilitated by the establishment of different animal model organisms, from flies to mammals. Here we describe the emerging model system of a Caenorhabditis elegans polymodal neuron named PVD, whose dendritic tree follows a stereotypical structure characterized by repeating candelabra-like structural units. In the past decade, progress has been made in understanding PVD's functions, morphogenesis, regeneration, and aging, yet many questions still remain. Expected final online publication date for the Annual Review of Neuroscience Volume 42 is July 8, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Wang L et al. (2023) STAR Protoc "Protocols for treating C. elegans with pharmacological agents, osmoles, and salts for imaging and behavioral assays."

Wang L, Bianchi L, Graziano B

[STAR Protoc, 2023]

Research rigor can be enhanced by pairing genetic tools with pharmacology and manipulations of solutes or ions. Here, we present a protocol for treating C. elegans with pharmacological agents, osmoles, and salts. We describe steps for agar plate supplementation, addition of the compound to the polymerized plates, and using liquid culture for exposure to the chemical. Treatment type depends on the stability and solubility of each compound. This protocol is applicable to both behavioral and in vivo imaging experiments. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022),¹ Fernandez-Abascal et al. (2022),² and Johnson et al. (2020).³.

Kim H et al. (2018) Mol Neurobiol "The Small GTPase RAC1/CED-10 Is Essential in Maintaining Dopaminergic Neuron Function and Survival Against -Synuclein-Induced Toxicity."

Caldwell GA, Kim H, Fernandez-Santiago R, Ezquerra M, Raya A, Carballo-Carbajal I, Consiglio A, Fernandez-Carasa I, Vila M, Dalfo E, Lizcano JM, Berkowitz L, Guha S, Miranda-Vizuete A, Calatayud C, Caldwell KA, Kapahi P

[Mol Neurobiol, 2018]

Parkinson's disease is associated with intracellular -synuclein accumulation and ventral midbrain dopaminergic neuronal death in the Substantia Nigra of brain patients. The Rho GTPase pathway, mainly linking surface receptors to the organization of the actin and microtubule cytoskeletons, has been suggested to participate to Parkinson's disease pathogenesis. Nevertheless, its exact contribution remains obscure. To unveil the participation of the Rho GTPase family to the molecular pathogenesis of Parkinson's disease, we first used C elegans to demonstrate the role of the small GTPase RAC1 (ced-10 in the worm) in maintaining dopaminergic function and survival in the presence of alpha-synuclein. In addition, ced-10 mutant worms determined an increase of alpha-synuclein inclusions in comparison to control worms as well as an increase in autophagic vesicles. We then used a human neuroblastoma cells (M17) stably over-expressing alpha-synuclein and found that RAC1 function decreased the amount of amyloidogenic alpha-synuclein. Further, by using dopaminergic neurons derived from patients of familial LRRK2-Parkinson's disease we report that human RAC1 activity is essential in the regulation of dopaminergic cell death, alpha-synuclein accumulation, participates in neurite arborization and modulates autophagy. Thus, we determined for the first time that RAC1/ced-10 participates in Parkinson's disease associated pathogenesis and established RAC1/ced-10 as a new candidate for further investigation of Parkinson's disease associated mechanisms, mainly focused on dopaminergic function and survival against -synuclein-induced toxicity.

McGhee JD et al. (2000) West Coast Worm Meeting "Looking for synergy with PHA-4 on the myo-2 promoter"

Okkema PG, McGhee JD, Kalb JM

[West Coast Worm Meeting, 2000]

PHA-4 is a fork head transcription factor that is essential for pharyngeal development. Forced expression of PHA-4 outside of the pharynx can activate pharynx-specific genes. For example, it has been previously shown that PHA-4 activates myo-2, pharyngeal myosin, through the C-subelement in the myo-2 promoter. Ectopic PHA-4 expression leads to strong ectopic expression of a C-subelement regulated reporter gene. However, it only weakly activates endogenous myo-2; with ectopic expression of PHA-4 throughout the embryo, ectopic expression of myo-2 is only detected in body wall muscles, showing factors in addition to PHA-4 are necessary for myo-2 expression. Thus, we suggest that PHA-4 works with co-factors to activate gene transcription. One likely candidate to be a co-factor with PHA-4 is PEB-1. PEB-1 is a novel DNA binding protein expressed in the pharynx and was identified by its ability to bind to the C-subelement in the myo-2 promoter (Thatcher, Fernandez, Beaster-Jones, Haun and Okkema). The binding sites for PHA-4 and PEB-1 overlap in the C-subelement. To test if PHA-4 and PEB-1 act synergistically via the C-subelement to activate gene expression, we have been assembling the transcriptional regulatory network in yeast. We find that both PHA-4 and PEB-1 are (rather weak) transcriptional activators on their own. However, when both PHA-4 and PEB-1 are expressed together, we find no synergism, either positive or negative. To continue our investigation into the biochemical basis of PHA-4 action, we have produced all three forms of PHA-4 in baculovirus to define preferred binding sites.

Kim H et al. (2018) Mol Neurobiol "Correction to: The Small GTPase RAC1/CED-10 Is Essential in Maintaining Dopaminergic Neuron Function and Survival Against α-Synuclein-Induced Toxicity."

Kapahi P, Vila M, Berkowitz L, Caldwell KA, Lizcano JM, Dalfo E, Calatayud C, Guha S, Caldwell GA, Kim H, Consiglio A, Ezquerra M, Carballo-Carbajal I, Raya A, Miranda-Vizuete A, Fernandez-Santiago R, Fernandez-Carasa I

[Mol Neurobiol, 2018]

With the author(s)' decision to opt for Open Choice the copyright of the article changed on March 2018 to

VACHON, C. et al. (2017) International Worm Meeting "Analysis of syndecan function at the C. elegans neuromuscular junction."

BULOW, H.E, TANG, L., VACHON, C., BESSEREAU, J-L.

[International Worm Meeting, 2017]

The extracellular matrix (ECM) plays an essential role in the development and function of organs and tissues. Syndecan is an ECM component that belongs to the heparan sulfate proteoglycan (HSPG) family. It is composed of 3 polysaccharidic chains linked to a core transmembrane protein. In C. elegans syndecan is coded by a single gene sdn-1. It is required for normal vulva development and axonal guidance (Minniti et al., 2004) (Rhiner et al., 2005). Sugar chains are extensively modified including sulfation, acetylation and epimerisation of individual sugar residues. Recent reports suggest that some modifications are cell specific (Attreed et al., 2016) supporting the hypothesis of an HS code. HSPG are present at neuromuscular junctions (NMJ) but their synaptic functions remain uncharacterized. In C. elegans body-wall muscle cell receive excitatory and inhibitory innervation from cholinergic and GABAergic motoneurons, respectively. Ce-punctin / MADD-4 is an ECM protein secreted by motoneurons in the synaptic cleft. Specific combinations of Ce-punctin isoforms trigger the clustering of acetylcholine or GABAA receptors at synaptic sites. We generated a BFP knock-in allele to detect SDN-1 and observed that SDN-1 is present at NMJs and seems to be enriched at cholinergic neuro-muscular synapses. Using single-chain antibodies to label specific HS modifications in vivo suggests that some modifications could be prevalent at cholinergic junctions. Preliminary data indicate that sdn-1 disruption affects the localization of acetylcholine receptors. Ongoing experiments and future plans will be presented at the meeting. Attreed, M., Saied-Santiago, K., Bulow, H.E., 2016. Conservation of anatomically restricted glycosaminoglycan structures in divergent nematode species. Glycobiology 26, 862-870. doi:10.1093/glycob/cww037 Minniti, A.N., Labarca, M., Hurtado, C., Brandan, E., 2004. Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva. J. Cell Sci. 117, 5179-5190. doi:10.1242/jcs.01394 Rhiner, C., Gysi, S., Frohli, E., Hengartner, M.O., Hajnal, A., 2005. Syndecan regulates cell migration and axon guidance in C. elegans. Development 132, 4621-4633. doi:10.1242/dev.02042

Gabaldon, C. et al. (2019) International Worm Meeting "Carnobacterium inhibits a bacterium innocuous for its fish natural host kills Caenorhabditis elegans."

Calixto, A., Garcia, V., Gabaldon, C.

[International Worm Meeting, 2019]

The Carnobacterium genus contains nine ubiquitous species of lactic acid bacteria isolated from cold to temperate environments (Leisner et al., 2007). The species Carnobacterium inhibens is a motile gram-positive bacterium first isolated from the intestine of the Atlantic Salmon (Salmo salar). C. inhibens inhibits bacterial growth of pathogenic Vibrio anguillarum and Aeromonas salmonicida by producing bacteriocins and antimicrobial peptides. Our group isolated a C. inhibens strain from a sample of fresh water of the Mapocho River in the city of Santiago de Chile with capacity to inhibit various clinically relevant bacterial pathogens in vitro. Nonetheless, safety of this strain has not been assessed. In this work we studied the effect of feeding the Chilean isolate C. inhibens to C. elegans. We evaluated the worm behavior and development while feeding on C. inhibens. Within the first hours animals develop major body paralysis with largely reduced pharynx pumping. 100% of L4 worms died 24 hours after exposure to C. inhibens. Strikingly, avoidance behavior in this C. inhibens strain develops much faster than in any other pathogenic bacteria reported, showing 25% of avoidance during the first 10 minutes and complete avoidance after 24 hours. The molecule that affects C. elegans is not volatile, because when constructing a plate with C. inhibens containing a smaller plate in its interior with worms fed with Escherichia coli OP50 and sharing the same environment that the toxic bacteria, these worms did not suffer any damage. This suggested that the toxic molecule needs to be ingested. To test this hypothesis, we exposed 1% SDS resistant daf-2 dauers at 25 deg C to C. inhibens. All dauers survived, indicating that the bacteria need to be eaten to cause its effect. All animals died after recovering from dauer. This suggests that this C. inhibits strain may not be innocuous for animal hosts and offers a platform for studying the molecules involved in the early response of the worm to pathogenic bacteria and prompts the identification of the bacterial molecules involved in the killing and their mechanism.

Nassar, Layla et al. (2015) International Worm Meeting "Understanding how sex modulates the female nervous system to drive distinct reproductive behavior states."

Collins, Kevin, Nassar, Layla, Bode, Addys

[International Worm Meeting, 2015]

We are interested in understanding how mating and reproductive behaviors are coordinated in the female nervous system. Specifically, we are identifying the neural signaling systems that drive two mutually exclusive vulval motor behaviors: mating with males or the release of progeny during egg laying [1]. We hypothesize that: 1. female vulval muscle twitching contractions facilitate male spicule insertion during mating; 2. specific mechanical and chemical signals report successful copulation and insemination; and 3. mating initiates reproductive behaviors including oocyte production and egg release. To investigate these hypotheses, we are using calcium imaging techniques to record vulval signaling events during mating with males, after successful insemination, and during the resumption of normal egg laying behavior. We have found that hermaphrodites with sperm have reduced vulval muscle twitching behaviors resulting in inefficient mating. In contrast, hermaphrodites depleted of sperm have increased vulval muscle twitching that facilitates male spicule insertion and mating. After mating is complete, hermaphrodites have sustained vulval muscle twitching that can result in release of sperm from the uterus. This behavior may act as a mechanism for competition with self-sperm. We are now examining how the other cells in the egg-laying circuit, including the HSN and VC neurons and uv1 neuroendocrine cells, respond during steps of male mating. During egg laying behavior, we found that the uv1 cells are mechanically activated by passage of eggs through the vulva, driving release of tyramine that inhibits egg laying [2]. We predict that mechanical stimulation by male spicules may similarly activate the uv1 cells to inhibit egg release during mating. However, once mating is complete, we expect egg laying to resume or even increase. Together, these results will explain how internal and external signals modulate activity in the same neural circuit to drive distinct behavior states.1. Collins, KM, and Koelle, MR (2013). Postsynaptic ERG Potassium Channels Limit Muscle Excitability to Allow Distinct Egg-Laying Behavior States in Caenorhabditis elegans. J. Neurosci. 33, 761-775.2. Collins KM, Bode A, Fernandez R, Tanis JE, Creamer M, Koelle MR (2015). Unpublished results.