Gao S et al. (2018) Elife "Excitatory motor neurons are local oscillators for backward locomotion."

Huang YC, Li Y, Jin Y, Lu Y, Zhen M, Guan SA, Alcaire S, Meng J, Alkema M, Hung W, Fouad AD, Gao S, Qi YB, Fang-Yen C, Kawano T

[Elife, 2018]

Cell- or network-driven oscillators underlie motor rhythmicity. The identity of C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron's oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron's intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.

Gao S et al. (2009) Cell Calcium "Mechanism of different spatial distributions of Caenorhabditis elegans and human STIM1 at resting state."

Xu T, Xu P, Lu J, Fan Y, Gao S, Chen L

[Cell Calcium, 2009]

Recent studies have identified STIM1 and Orai1 as essential and conserved components of the Ca(2+) release-activated Ca(2+) (CRAC) channel. However, the reason STIM1 exhibits different distributions in nematode Caenorhabditis elegans and in human cells before endoplasmic reticulum (ER) calcium store depletion has not been clarified. Compared to the diffuse ER distribution of human STIM1 (H.STIM1), we found that C. elegans STIM1 (C.STIM1) was pre-oligomerized in puncta at the cell periphery before Ca(2+) store depletion when expressed in HEK293 cells. Interestingly, these C.STIM1 puncta failed to induce aggregation of C. elegans Orai1 (C.Orai1), and no CRAC current was detected in quiescent cells. However, upon store depletion, C.Orai1 and C.STIM1 functioned as a pair to locally sense the store depletion signal and to activate the CRAC channel. We substituted the N-terminus of H.STIM1 for the N-terminus of C.STIM1 (H_C.STIM1), which resulted in pre-puncta resting localization. In contrast, by replacing the C-terminus of C.STIM1 with that of H.STIM1, we made a chimeric protein (C.STIM1_H) that exhibited two distribution profiles at resting state, a diffuse ER pattern like H.STIM1, and large aggregates. Taken together, our results suggest that (1) despite highly conserved functional domains, C. elegans STIM1 and human STIM1 display different spatial distributions in HEK293 cells before store depletion; (2) the C.STIM1 puncta at peripheral sites are not sufficient for the aggregation and activation of C.Orai1 in the absence of store depletion; (3) the distinct distributions of C.STIM1 and H.STIM1 at resting state are determined by the cytoplasmic region of STIM1.

Gao S et al. (2015) Nat Commun "The NCA sodium leak channel is required for persistent motor circuit activity that sustains locomotion."

Guan S, Gao S, Po MD, Zhen M, Kawano T, Xie L

[Nat Commun, 2015]

Persistent neural activity, a sustained circuit output that outlasts the stimuli, underlies short-term or working memory, as well as various mental representations. Molecular mechanisms that underlie persistent activity are not well understood. Combining in situ whole-cell patch clamping and quantitative locomotion analyses, we show here that the Caenorhabditis elegans neuromuscular system exhibits persistent rhythmic activity, and such an activity contributes to the sustainability of basal locomotion, and the maintenance of acceleration after stimulation. The NALCN family sodium leak channel regulates the resting membrane potential and excitability of invertebrate and vertebrate neurons. Our molecular genetics and electrophysiology analyses show that the C. elegans NALCN, NCA, activates a premotor interneuron network to potentiate persistent motor circuit activity and to sustain C. elegans locomotion. Collectively, these results reveal a mechanism for, and physiological function of, persistent neural activity using a simple animal model, providing potential mechanistic clues for working memory in other systems.

Gao S et al. (2011) Proc Natl Acad Sci U S A "Action potentials drive body wall muscle contractions in Caenorhabditis elegans."

Zhen M, Gao S

[Proc Natl Acad Sci U S A, 2011]

The sinusoidal locomotion exhibited by Caenorhabditis elegans predicts a tight regulation of contractions and relaxations of its body wall muscles. Vertebrate skeletal muscle contractions are driven by voltage-gated sodium channel-dependent action potentials. How coordinated motor outputs are regulated in C. elegans, which does not have voltage-gated sodium channels, remains unknown. Here, we show that C. elegans body wall muscles fire all-or-none, calcium-dependent action potentials that are driven by the L-type voltage-gated calcium and Kv1 voltage-dependent potassium channels. We further demonstrate that the excitatory and inhibitory motoneuron activities regulate the frequency of action potentials to coordinate muscle contraction and relaxation, respectively. This study provides direct evidence for the dual-modulatory model of the C. elegans motor circuit; moreover, it reveals a mode of motor control in which muscle cells integrate graded inputs of the nervous system and respond with all-or-none electrical signals.

Gao S et al. (2018) BMC Pharmacol Toxicol "Classification and prediction of toxicity of chemicals using an automated phenotypic profiling of Caenorhabditis elegans."

Xian B, Jois M, Jing H, Zeng Y, Han JJ, Zhang N, Gao S, Flavel M, Li G, Chen W

[BMC Pharmacol Toxicol, 2018]

BACKGROUND: Traditional toxicological studies have relied heavily on various animal models to understand the effect of various compounds in a biological context. Considering the great cost, complexity and time involved in experiments using higher order organisms. Researchers have been exploring alternative models that avoid these disadvantages. One example of such a model is the nematode Caenorhabditis elegans. There are some advantages of C. elegans, such as small size, short life cycle, well defined genome, ease of maintenance and efficient reproduction. METHODS: As these benefits allow large scale studies to be initiated with relative ease, the problem of how to efficiently capture, organize and analyze the resulting large volumes of data must be addressed. We have developed a new method for quantitative screening of chemicals using C. elegans. 33 features were identified for each chemical treatment. RESULTS: The compounds with different toxicities were shown to alter the phenotypes of C. elegans in distinct and detectable patterns. We found that phenotypic profiling revealed conserved functions to classify and predict the toxicity of different chemicals. CONCLUSIONS: Our results demonstrate the power of phenotypic profiling in C. elegans under different chemical environments.

Gao S et al. (2015) Nat Commun "Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp."

Schneider MW, Kozjak-Pavlovic V, Gao S, Nagel G, Gottschalk A, Nagpal J

[Nat Commun, 2015]

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20C) 17 cGMPs(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Gao S et al. (2019) J Vis Exp "A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans."

Zhang W, Xian B, Jing H, Zhang N, Han JJ, Li G, Flavel M, Xu C, Chen W, Han G, Gao S, Zeng Y, Jois M

[J Vis Exp, 2019]

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

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.