Ke T et al. (2020) Curr Protoc Toxicol "Generating Bacterial Foods in Toxicology Studies with Caenorhabditis elegans."

Santamaria A, Bornhorst J, Aschner M, Ke T, Tinkov AA

[Curr Protoc Toxicol, 2020]

Caenorhabditis elegans is a free-living animal that is used as a powerful experimental model in biological sciences. The natural habitat of the animal are areas rich in material from rotting plants or fruits being decomposed by a growing number of microorganisms. The ecology of the natural habitat of C. elegans is a complex interactive network involving many species, including numerous types of bacteria, viruses, fungi, slugs, snails, and isopods, among which bacteria play multifaceted roles in the natural history of C. elegans. Under laboratory conditions, C. elegans is routinely cultured in a petri dish filled with solidified agar and seeded with Escherichia coli strain OP50, the latter offering an alternative model to study the interaction between bacteria and host. Because of the clear advantages of generating specific bacterial foods for mechanistic studies in C. elegans, it is important to develop a robust protocol to generate high-quality bacterial foods commensurate with experimental requirements. Based on previous work by us and others, herein we present a protocol on how to generate these optimal bacterial food-based research tools. 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparing concentrated E. coli OP50 Basic Protocol 2: Titrating bacteria concentration Basic Protocol 3: Generating dead bacterial food by heating Basic Protocol 4: Generating dead bacterial food by antibiotics Basic Protocol 5: Feeding C. elegans with bacterial foods in liquid.

Ke T et al. (2021) Neurochem Res "The Role of Human LRRK2 in Acute Methylmercury Toxicity in Caenorhabditis elegans."

Santamaria A, Tinkov AA, Aschner M, Rocha JBT, Bowman AB, Ke T

[Neurochem Res, 2021]

Methylmercury (MeHg) exposure and its harmful effects on the developing brain continue to be a global environmental health concern. Decline in mitochondrial function is central to the toxic effects of MeHg and pathogenesis of mitochondria-related diseases including Parkinson's disease (PD). LRRK2 (Leucine-rich repeat kinase 2) mutation is one of the most common genetic risk factors for PD. In this study, we utilize an acute toxicity model of MeHg exposure in the model organism Caenorhabditis elegans (C. elegans) to compare lifespan, developmental progression, mitochondrial membrane potential and reactive oxygen species (ROS) between the wild-type N2 strain, wild-type LRRK2 transgenic strain (WLZ1), and mutant LRRK2(G2019S) transgenic strain (WLZ3). Additionally, the expression levels of skn-1 and gst-4 were investigated. Our results show that acute MeHg exposure (5 and 10M) caused a significant developmental delay in the N2 and WLZ3 worms. Notably, the worms expressing wild-type LRRK2 were resistant to 5M MeHg- induced developmental retardation. ROS levels in response to MeHg exposure were increased in the N2 worms, but not in the WLZ1 or WLZ3 worms. The mitochondrial membrane potential was decreased in the N2 worms but increased in the WLZ1 and WLZ3 worms following MeHg exposure. Furthermore, MeHg exposure increased the expression of skn-1 in N2, but not in WLZ1 worms. Although skn-1 expression was increased in the WLZ3 worms following MeHg exposure, gst-4 expression was not induced. Both skn-1 and gst-4 had higher basal expression levels in LRRK2s transgenic than wild-type N2 worms. Knocking down of skn-1 with feeding RNAi had a significant developmental effect in WLZ1 worms; however, the effect was not found in WLZ3 worms. These results suggest that mitochondrial dysfunction and a defect in the SKN-1 signaling in the LRRK2 G2019S worms contribute to the severe developmental delay, establishing a modulatory role of LRRK2 mutation in MeHg-induced acute toxicity.

Ke T et al. (2020) Toxicol Rep "N,N' bis-(2-mercaptoethyl) isophthalamide induces developmental delay in Caenorhabditis elegans by promoting DAF-16 nuclear localization."

Antunes Soares FA, Skalny AV, Santamaria A, Aschner M, Bowman AB, Ke T

[Toxicol Rep, 2020]

N,N' bis-(2-mercaptoethyl) isophthalamide (NBMI) is a lipophilic thiol-containing agent that has high affinity for toxic metal ions, such as Hg²⁺, Pb²⁺, and Cd²⁺. Studies have shown that NBMI is a potent chelator of heavy metals, yet its potential toxicity in animals has yet to be determined. Using the model organism <i>Caenorhabditis elegans</i> (<i>C. elegans</i>), we show no significant change in worms' death rate or lifespan following NBMI treatment (10-1000 M). However, NBMI treatment was associated with a significant developmental delay. To determine if the <i>daf-2/age-1/daf-16</i> pathway is involved in NBMI toxicity, mRNA levels of these genes were assessed in worms treated with NBMI. Here, we found that while NBMI failed to significantly alter the expression of <i>daf-16</i> or <i>daf-2</i>; <i>age-1</i> was significantly downregulated by NBMI. Furthermore, NBMI significantly increased DAF-16 nuclear localization. Consistent with a role for this pathway in NBMI toxicity, the developmental arrest by NBMI was more prominent in the DAF-16 transgenic strain than in the wild type N2 strain. Moreover, in the mutant strains harboring null alleles of <i>daf-16</i>, NBMI had no effect on development. In addition, NBMI repressed the expression of detoxifying genes (<i>skn-1, gst-4</i> and <i>gcs-1</i>). In summary, NBMI has a low developmental toxicity in the <i>C. elegans</i> model, and the nuclear translocation of DAF-16 is involved in the developmental effect of NBMI.

Ke T et al. (2022) Neurotox Res "The Modulatory Role of sti-1 in Methylmercury-Induced Toxicity in Caenorhabditis elegans."

Aschner M, Bowman AB, Rocha JBT, Farina M, Ke T, Santamaria A

[Neurotox Res, 2022]

Human exposure to the neurotoxin methylmercury (MeHg) poses a significant health risk to the development of the nervous system. The mechanisms of MeHg-induced neurotoxicity are associated with the disruption of cellular homeostasis, and include oxidative stress, loss of calcium homeostasis, and impaired protein quality control. The stress inducible protein 1 (STI-1) is involved in the regulation of protein quality control by acting as a protein cochaperone to maintain optimal protein unfolding and refolding. Here, we utilized the Caenorhabditis elegans (C. elegans) model of MeHg toxicity to characterize the role of the sti-1 gene in MeHg-induced toxicity. We showed that lifespan and developmental milestone timings were significantly altered in sti-1 knockout (KO) animals with MeHg exposure. However, knocking down sti-1 by RNAi did not result in an analogous effect for lifespan, but did still sensitize to delays in developmental milestone progression by acute MeHg, suggesting that insufficiency of sti-1 does not recapitulate all phenotypes of the null mutation. Furthermore, inhibition of ATP levels by MeHg exposure was modulated by sti-1. Considering that the skn-1/gst-4 pathway is highly involved in metal's toxicity, such pathway was also explored in our model. We showed that sti-1 mutant worms exhibited impaired capacity to upregulate the antioxidant genes skn-1/gst-4, highlighting a central role of sti-1 in modulating antioxidant response. Lastly, we showed that loss-of-function mutation in the rrf-3 gene, which encodes a putative RNA-directed RNA polymerase, has significant effect in altering MeHg-induced toxicity by potentiating the animal's detoxification system. Altogether, our novel data show an indispensable role of protein quality control in the defense against MeHg toxicity.

Ke T et al. (2022) Neurotoxicol Teratol "Methylmercury exposure-induced reproductive effects are mediated by dopamine in Caenorhabditis elegans."

Rocha JBT, Aschner M, Junior FB, Bowman AB, Santamaria A, Ke T

[Neurotoxicol Teratol, 2022]

Methylmercury (MeHg) is a neurotoxicant that exists in the natural environment, which level can be greatly increased because of human activity. MeHg exposures have the risk of being detrimental to the development of the nervous system. Studies on MeHg toxicity have largely focused on the mechanisms of its neurotoxicity following developmental exposures. Additionally, reproductive toxicity of developmental MeHg exposures has been noted in rodent models. The model organism Caenorhabditis elegans (C. elegans) is a self-fertilizing animal which has a short lifespan around 20 days. Most C. elegans are hermaphrodites that can generate both sperm and oocytes. To investigate the effects of developmental MeHg exposures on the reproduction in C. elegans, larvae stage 1 worms were exposed to MeHg (0, 0.01 or 0.05 &#956;M) for 24 h. The laid eggs and oocytes were compared during each day at adult stages for 6 days. We showed that MeHg exposure significantly induced an increased number of eggs in day 1 adults without an effect on the timing of egg laying or the total number of eggs or oocytes over the 6-day period. The expression of dat-1 and cat-2 and dopamine levels were increased in worms exposed to MeHg. Supplementation with 100 &#956;M dopamine recapitulated the effect of MeHg on the number of eggs present in day 1 adults. Furthermore, the effect of MeHg on the number of eggs was abrogated in the cat-2 mutant worms CB1112. The number of oocytes in the 6-day adult stages was decreased by MeHg in the dat-1 mutant RM2702. MeHg exposures did not change the mating rate or the number of offspring from mating. Combined, these novel findings show that developmental exposure to low levels of MeHg has limited effects on the reproduction in C. elegans. Furthermore, our data support a modulatory role of dopamine in MeHg-induced effects on reproduction in this model system.

Parrilla A et al. (2016) Cell Cycle "Mitotic entry: The interplay between Cdk1, Plk1 and Bora."

Gotta M, Cirillo L, Parrilla A, Santamaria A, Thomas Y, Pintard L

[Cell Cycle, 2016]

Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase. In a recent article published in Cell Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1 activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp residues that are located in the N-terminal, most conserved part, of SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1 function in mitotic entry in C. elegans embryos and during DNA damage checkpoint recovery in mammalian cells. Here, using an untargeted Forster Resonance Energy Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional experimental evidence supporting the importance of these phosphorylation sites for Plk1 activation and subsequent mitotic entry after DNA damage. We also briefly discuss the mechanism of Plk1 activation and the potential role of Bora phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer cells and this correlates with poor prognosis, understanding how Bora contributes to Plk1 activation is paramount for the development of innovative therapeutical approaches.

Estrada-Valencia R et al. (2021) Rev Physiol Biochem Pharmacol "The Endocannabinoid System in Caenorhabditis elegans."

Rangel-Lopez E, Santamaria A, Colonnello A, Aschner M, Saraiva NR, de Lima ME, de Avila DS, Estrada-Valencia R

[Rev Physiol Biochem Pharmacol, 2021]

The existence of a formal Endocannabinoid System in C. elegans has been questioned due to data showing the absence of typical cannabinoid receptors in the worm; however, the presence of a full metabolism for endocannabinoids, alternative ligands, and receptors for these agents and a considerable number of orthologous and homologous genes regulating physiological cannabinoid-like signals and responses - several of which are similar to those of mammals - demonstrates a well-structured and functional complex system in nematodes. In this review, we describe and compare similarities and differences between the Endocannabinoid System in mammals and nematodes, highlighting the basis for the integral study of this novel system in the worm.

Chan YW et al. (2009) J Cell Biol "Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly."

Schmitz MH, Chan YW, Gerlich DW, Nigg EA, Santamaria A, Fava LL, Uldschmid A

[J Cell Biol, 2009]

Mitotic spindle formation and chromosome segregation depend critically on kinetochore-microtubule (KT-MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation.

Ke T et al. (2020) Neurochem Res "Cephalic Neuronal Vesicle Formation is Developmentally Dependent and Modified by Methylmercury and sti-1 in Caenorhabditis elegans."

Rocha JBT, Tinkov A, Santamaria A, Bowman AB, Bornhorst J, Ke T, Aschner M

[Neurochem Res, 2020]

Methylmercury (MeHg) is a potent neurotoxicant. The mechanisms underlying MeHg-induced neurotoxicity are not fully understood. Several studies have shown that protein chaperones are involved in MeHg toxicity. The protein co-chaperone, stress inducible protein 1 (STI-1), has important functions in protein quality control of the chaperone pathway. In the current study, dopaminergic (DAergic) cephalic (CEP) neuronal morphology was evaluated in the Caenorhabditis elegans (C. elegans) sti-1 knockout strain. In the control OH7193 strain (dat-1::mCherry+ttx-3::mCherry), we characterized the morphology of CEP neurons by checking the presence of attached vesicles and unattached vesicles to the CEP dendrites. We showed that the attached vesicles were only present in adult stage worms; whereas they were absent in the younger L3 stage worms. In the sti-1 knockout strain, MeHg treatment significantly altered the structures of CEP dendrites with discontinuation of mCherry fluorescence and shrinkage of CEP soma, as compared to the control. 12 h post treatment on MeHg-free OP50-seeded plates, the discontinuation of mCherry fluorescence of CEP dendrites in worms treated with 0.05 or 0.5M MeHg returned to levels statistically indistinguishable from control, while in worms treated with 5M MeHg a higher percentage of discontinuation of mCherry fluorescence persisted. Despite this strong effect by 5M MeHg, CEP attached vesicles were increased upon 0.05 or 0.5M MeHg treatment, yet unaffected by 5M MeHg. The CEP attached vesicles of sti-1 knockout strain were significantly increased shortly after MeHg treatment, but were unaffected 48h post treatment. In addition, there was a significant interactive effect of MeHg and sti-1 on the number of attached vesicles. Knock down sti-1 via RNAi did not alter the number of CEP attached vesicles. Taking together, our data suggests that the increased occurrence of attached vesicles in adult stage worms could initiate a substantial loss of membrane components of CEP dendrites following release of vesicles, leading to the discontinuation of mCherry fluorescence, and the formation of CEP attached vesicles could be regulated by sti-1 to remove cellular debris for detoxification.

da Silveira TL et al. (2018) Neurotoxicology "Quinolinic Acid and glutamatergic neurodegeneration in Caenorhabditis elegans."

Camara DF, Santamaria A, Zamberlan DC, Soares FAA, Machado ML, Aschner M, da Silva TC, Arantes LP, da Silveira TL

[Neurotoxicology, 2018]

Quinolinic acid (QUIN) is an endogenous neurotoxin that acts as an N-methyl-D-aspartate receptor (NMDAR) agonist generating a toxic cascade, which can lead to neurodegeneration. The action of QUIN in Caenorhabditis elegans and the neurotoxins that allow the study of glutamatergic system disorders have not been carefully addressed. The effects of QUIN on toxicological and behavioral parameters in VM487 and VC2623 transgenic, as well as wild-type (WT) animals were performed to evaluate whether QUIN could be used as a neurotoxin in C. elegans. QUIN reduced survival of WT worms in a dose-dependent manner. A sublethal dose of QUIN (20mM) increased reactive oxygen species (ROS) levels in an nmr-1/NMDAR-dependent manner, activated the DAF-16/FOXO transcription factor, and increased expression of the antioxidant enzymes, superoxide dismutase-3, glutathione S-transferase-4, and heat shock protein-16.2. QUIN did not change motor behavioral parameters, but altered the sensory behavior in N2 and VM487 worms. Notably, the effect of QUIN on the sensory behavioral parameters might occur, at least in part, secondary to increased ROS. However, the touch response behavior indicates a mechanism of action that is independent of ROS generation. In addition, non-lethal doses of QUIN triggered neurodegeneration in glutamatergic neurons. Our findings indicate that C. elegans might be useful as a model for studies of QUIN as a glutamatergic neurotoxin in rodent models.