Prigent Garcia, S. et al. (2019) International Worm Meeting "Understanding kinetic delays in biological systems."

Prigent Garcia, S., Begnaud, S., Bourdon, L., Suman, S., Plancke, C., Robin, F.

[International Worm Meeting, 2019]

Cascades of activation are defined by a succession of sequential activation of signaling proteins. This leads to the activation of a downstream effector, typically affecting a specific biological function with a precise intensity and timing. Here, we used the RhoA activation cascade as a model to analyse the unfolding of this cascade in a cell. In this cascade, we can measure, at a specific location of the cell cortex, a stereotypical delay between the activation of the upstream regulator and the recruitment and activation of the downstream effector. First, we proceeded to a careful caracterization of the dynamics of two sequential steps of the cascade. Using TIRF microscopy, we focused on the different steps of the RhoA/ROCK/Myosin and RhoA/Formin/Actin activation cascades, using the Myosin as a landmark to measure the delays within the cascade. Second, using single-molecule imaging, we focused on the last step of this cascade and measured the dynamic modulation of the binding (Kon) and the unbinding rate (Koff) of the Myosin at the cortex of C. elegans early embryos. We then developed a simple numerical model that takes advantage of the dynamic measurements of Kon and Koff to predict the temporal evolution of this cascade. We propose that this simple and generic model - which can in essence fit any activation cascade - offers a simple mathematical framework to understand the temporal dynamics of signaling cascades, and the delay and change in the shape of the response which can be observed between the input and the output of a cascade.

Garcia, S. et al. (2015) International Worm Meeting "Identification of trinucleotide-repeat RNA toxicity pathways in C. elegans."

Ruvkun, G., Tabach, Y., Armakola, M., Garcia, S., Lourenco, G.

[International Worm Meeting, 2015]

Myotonic dystrophy disorders are caused by expanded CUG repeats in noncoding regions. We used Caenorhabditis elegans expressing CUG repeats to identify genes that modulate the toxicity of such repeats. We identified 15 conserved genes that function as suppressors or enhancers of CUG repeat-induced toxicity and that modulate formation of nuclear foci by CUG-repeat RNA. These genes regulate CUG repeat-induced toxicity through distinct mechanisms including RNA export and clearance, thus supporting a more complex regulation of RNA toxicity than previously thought where multiple pathways mediate CUG-repeat toxicity. A subset of the genes identified is also involved in other degenerative disorders.We find that the nonsense-mediated mRNA decay (NMD) pathway has a conserved role in regulating CUG-repeat-RNA transcript levels and toxicity. In addition, NMD recognition of toxic RNAs depends on the 3'-untranslated-region GC-nucleotide content. Our studies suggest a broader surveillance role for NMD in which variations in this pathway influence multiple degenerative diseases.

Hernandez-Garcia S et al. (2024) Food Funct "Olive oil tyrosols reduce α-synuclein aggregation in vitro and in vivo after ingestion in a Caenorhabditis elegans Parkinson's model."

Hernandez-Garcia S, Henarejos-Escudero P, Martinez-Rodriguez P, Garcia-Cano B, Gandia-Herrero F

[Food Funct, 2024]

Parkinson's disease is the neurodegenerative motor disorder with the highest incidence worldwide. Among other factors, Parkinson's disease is caused by the accumulation of &#x3b1;-synuclein aggregates in a patient's brain. In this work, five molecules present in the diet are proposed as possible nutraceuticals to prevent and/or reduce the formation of &#x3b1;-synuclein oligomers that lead to Parkinson's disease. The olive oil polyphenols tyrosol, hydroxytyrosol (HT), hydroxytyrosol acetate (HTA) and dihydroxyphenyl acetic acid (DOPAC) besides vitamin C were tested using a cellular model of &#x3b1;-synuclein aggregation and a <i>Caenorhabditis elegans</i> Parkinson's disease animal model. Levodopa was included in the assays as the main drug prescribed to treat the disease as well as dopamine, its direct metabolite. HTA and DOPAC completely hindered &#x3b1;-synuclein aggregation <i>in vitro</i>, while dopamine reduced the aggregation by 28.7%. The Parallel Artificial Membrane Permeability Assay (PAMPA) showed that HTA had the highest permeability through brain lipids among the compounds tested. Furthermore, the <i>C. elegans</i> Parkinson's disease model made it possible to assess the chosen compounds <i>in vivo</i>. The more effective substances <i>in vivo</i> were DOPAC and HTA which reduced the &#x3b1;S aggregation inside the animals by 79.2% and 76.2%, respectively. Moreover, dopamine also reduced the aggregates by 67.4% in the <i>in vivo</i> experiment. Thus, the results reveal the potential of olive oil tyrosols as nutraceuticals against &#x3b1;-synuclein aggregation.

Vrbanac A et al. (2017) Cell Host Microbe "An Elegan(t) Screen for Drug-Microbe Interactions."

Morton JT, Debelius JW, Knight R, Jiang L, Dorrestein P, Vrbanac A

[Cell Host Microbe, 2017]

Microbes affect drug responses, but mechanisms remain elusive. Two papers in Cell exploit C.elegans to infer anticancer drug mechanisms. Through high-throughput screens of drug-microbe-host interactions, Garcia-Gonzalez etal. (2017) and Scott etal. (2017) determine that bacterial metabolism underpins fluoropyrimidine cytotoxicity, providing a paradigm for unraveling bacterial mechanisms in drug metabolism.

Teixeira, J. et al. (2019) International Worm Meeting "Identification of the ubiquinone pathway as a suppressor of Myotonic Dystrophy-associated toxicity."

Brandao, A., Garcia, S., Teixeira, J., Harju, A.

[International Worm Meeting, 2019]

Myotonic dystrophy type 1 (DM1) is the most common type of dystrophy in adulthood. It is caused by the accumulation of mutant RNA in the nucleus due to the expression of expanded CUG repeats in the 3' UTR of the myotonic dystrophy protein kinase (DMPK) gene. These RNA-bearing CUG repeats form hairpin structures that interact inappropriately to RNA binding proteins such as splicing factors, namely muscleblind-like (Mbnl) and CUG-binding protein (CUGBP) families, thereby causing aberrant alternative splicing leading to a multisystemic disease. Notably, due to the multisystemic nature of DM1, the full extent of cellular processes affected by these toxic RNAs is still unknown. Our aim is to identify new modulators and mechanisms of RNA toxicity. To address this aim, we performed an RNAi screen by using a previously characterized C. elegans DM1 model[1]. Since this C. elegans model mimics DM1 phenotypes, changes in its motility defect were used as readout for toxicity. We identified the ubiquinone (CoQ) pathway as a suppressor of DM1, as downregulation of this pathway increases DM1 toxicity, whereas CoQ supplementation partially rescues the DM1 motility defect. Furthermore, our data also suggest that complex II of mitochondrial electron transport chain is implicated in DM1 dysfunction. The role of mitochondria in DM1 pathogenesis is further underlined by our preliminary results showing that DM1 animals have an altered mitochondrial morphology. Taken together, we established a genetic connection between DM1, ubiquinone pathway and mitochondrial function and are currently examining the mechanisms of DM1 mitochondrial dysfunction regulation. 1. Garcia, S.M., et al., Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans. Nat Struct Mol Biol, 2014. 21(8): p. 712-20.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Vandecandelaere I et al. (2017) PLoS One "Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus."

Deforce D, Coenye T, Van Nieuwerburgh F, Vandecandelaere I

[PLoS One, 2017]

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.

Vandecandelaere I et al. (2014) Pathog Dis "Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms."

Coenye T, Vandecandelaere I, Depuydt P, Nelis HJ

[Pathog Dis, 2014]

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.

Rodrigues, Pedro Reis et al. (2009) International Worm Meeting "A variety of age-dependant aggregating proteins determine lifespan."

Peters, Theodore, Gibson, Brad, Lithgow, Gordon, Hughes, Robert, Alavez, Silvestre, Rodrigues, Pedro Reis, Czerwieniec, Gregg

[International Worm Meeting, 2009]

Protein aggregation has for long been hypothesised as a determinant of lifespan. Briefly, normal cellular activity may give rise to damaged proteins causing them to become insoluble, missfold and aggregate. To test this hypothesis we adapted a protocol in order to extract insoluble proteins from synchronously aging populations of C. elegans. Proteins were separated based on their aqueous and detergent solubility and the insoluble fraction was resolubilized in 70%; formic acid. Insoluble proteins were chemically labelled, identified and quantified by liquid chromatography coupled with mass spectrometry (LC- ESI-MS/MS). We identified a range of proteins with roles in various cellular processes and possibly from a range of cellular compartments. 27%; of the proteins identified as forming aggregates have previously been shown to be important in keeping low levels of polyglutamine aggregation1. This suggests that reduced soluble levels of these proteins caused by age-related aggregation may cause increased risk of polyglutamine aggregation. We then considered whether proteins that appear to form aggregates during normal aging influenced lifespan. To test this notion we, reduced their expression in adult animals (from 4 days old) by RNA interference (RNAi). 34%; of the RNAi treatments were found to significantly extend mean lifespan in C. elegans suggesting that a variety of age-dependant aggregating proteins determine lifespan. Among the insoluble proteins, DAF-21, an ortholog of the mammalian HSP-90, showed age-dependant aggregation and is being used as a marker to study the role of several molecular pathways in protein aggregation. Taken together our results suggest that protein aggregation may play a common and key role in aging and age-related disease. 1 - Nollen, E., Garcia, S., Haaften, G., Kim, S., Chavez, A., Morimoto, R., Plasterk, R., Genome-wide RNA interference screen identified previously undescribed regulators of polyglutamine aggregation. Proc Natl. Acad. Sci., 101, 6403-6408, 2004.