A Single Gene Target of an ETS-Family Transcription Factor Determines Neuronal CO2-Chemosensitivity

Many animals possess neurons specialized for the detection of carbon dioxide (CO2), which acts as a cue to elicit behavioral responses and is also an internally generated product of respiration that regulates animal physiology. In many organisms how such neurons detect CO2 is poorly understood. We report here a mechanism that endows C. elegans neurons with the ability to detect CO2. The ETS-5 transcription factor is necessary for the specification of CO2-sensing BAG neurons. Expression of a single ETS-5 target gene, gcy-9, which encodes a receptor-type guanylate cyclase, is sufficient to bypass a requirement for ets-5 in CO2-detection and transforms neurons into CO2-sensing neurons. Because ETS-5 and GCY-9 are members of gene families that are conserved between nematodes and vertebrates, a similar mechanism might act in the specification of CO2-sensing neurons in other phyla

IRK-1 Potassium Channels Mediate Peptidergic Inhibition of Caenorhabditis elegans Serotonin Neurons via a Gₒ Signaling Pathway

To identify molecular mechanisms that function in G-protein signaling, we have performed molecular genetic studies of a simple behavior of the nematode Caenorhabditis elegans, egg laying, which is driven by a pair of serotonergic neurons, the hermaphrodite-specific neurons (HSNs). The activity of the HSNs is regulated by the Gₒ-coupled receptor EGL-6, which mediates inhibition of the HSNs by neuropeptides. We report here that this inhibition requires one of three inwardly rectifying K+ channels encoded by the C. elegans genome: IRK-1. Using ChannelRhodopsin-2-mediated stimulation of HSNs, we observed roles for egl-6 and irk-1 in regulating the excitability of HSNs. Although irk-1 is required for inhibition of HSNs by EGL-6 signaling, we found that other Gₒ signaling pathways that inhibit HSNs involve irk-1 little or not at all. These findings suggest that the neuropeptide receptor EGL-6 regulates the potassium channel IRK-1 via a dedicated pool of Gₒ not involved in other Gₒ-mediated signaling. We conclude that G-protein-coupled receptors that signal through the same G-protein in the same cell might activate distinct effectors and that specific coupling of a G-protein-coupled receptor to its effectors can be determined by factors other than its associated G-proteins.National Institutes of Health (U.S.) (NIH Grant R01-GM024663)National Institutes of Health (U.S.) (NIH Grant R01-GM098320

Expression of the ETS-5 target gene <i>gcy-9</i> restores CO₂-chemosensitivity to <i>ets-5</i> mutants.

<p>(A) <i>gcy-36</i> and <i>gcy-18</i> promoters, which are active in oxygen-sensing and thermosensory neurons, respectively, are not regulated by ETS-5. Shown are lateral views of wild-type and <i>ets-5</i> mutant animals carrying either a <i>gcy-36</i> reporter, which is expressed by the oxygen-sensing URX neurons, or a <i>gcy-18</i> reporter, which is expressed by the thermosensory AFD neurons. A: anterior, V: ventral, scale bar is 20 µm. The <i>Prom_gcy-36::cameleon</i> transgene used was <i>wzEx39</i> and the <i>Prom_gcy-18::gfp</i> transgene was <i>wzEx40</i>. (B) Expression of <i>gcy-9</i> in either the URX oxygen sensors or the AFD thermosensors rescues the behavioral defect of <i>ets-5</i> mutants. The left plot shows the mean fraction of animals ± SEM that reversed during a four second exposure to either control atmosphere (0% CO₂, 20% O₂, balance N₂) or CO₂-enriched atmosphere (10% CO₂, 20% O₂, balance N₂). The right plot shows the effect of CO₂ on reversals as measured with an avoidance index, as in Fig. 2C. Plotted are the mean avoidance indices for each of the four strains tested ± SEM. P values were calculated by one-way ANOVA. <i>N</i> = 3–5 populations of 30–50 animals. The <i>ets-</i>5 mutant strain used was FX1734. The <i>Prom</i>_{gcy<i>-36</i>}<i>::gcy-9</i> transgene used was <i>wzIs97</i> and the <i>Prom_gcy-18::gcy-9</i> transgene was <i>wzEx34</i>. (C) Expression of <i>gcy-9</i> in the URX oxygen sensors confers sensitivity to CO₂. The ratiometric calcium indicator cameleon was expressed in the URX neurons. Wild-type URX neurons (top panel) showed a small decrease in the ratio of YFP:CFP emissions in response to 10 s CO₂ pulses indicating decreases in cell calcium. URX neurons expressing <i>gcy-9</i> showed increases in cell calcium in response to CO₂ stimuli, with an average ratio change of greater than 20% (lower panel). The cameleon expression transgene used was <i>wzIs96[Prom_gcy-32::cameleon]</i> and the <i>gcy-9</i> expression transgene was <i>wzIs97[Prom_gcy-36::gcy-9].</i> (D) Expression of <i>gcy-9</i> in the AFD thermosensors confers sensitivity to CO₂. Calcium responses of wild-type AFD neurons (top panel) and AFD neurons that express <i>gcy-9</i> (bottom panel) in responses to a 10% CO₂ stimulus. The cameleon expression transgene was <i>fxIs105[Prom_gcy-8::cameleon]</i>, and the <i>Prom_gcy-18::gcy-9</i> transgene was <i>wzEx34.</i> For panels C and D, plots are mean YFP/CFP emissions ratios normalized to the pre-stimulus ratio R₀ (<i>N</i> = 16–22 animals). Red shaded areas represent SEM.</p

An ETS-family transcription factor is required for the specification of <i>C. elegans</i> CO₂-chemosensitive BAG neurons.

<p>(A) A 31 basepair DNA element comprising a single ETS-binding motif (top) drives expression of GFP specifically in the BAG chemosensitive neurons (bottom). (B) One of ten ETS-family transcription factors encoded by the <i>C. elegans</i> genome is required for specification of BAG neurons. Shown is percent of animals mutant for each of ten ETS-family transcription factors encoded by the <i>C. elegans</i> genome that are BAGL/R ON (green circles) and BAGL/R OFF (open circles) for expression of a <i>Prom_flp-19::gfp</i> reporter transgene. <i>N</i> = number of animals scored. # We found one <i>lin-1(e1777)</i> mutant in which <i>Prom_flp-19::gfp</i> was not expressed in BAGR. (C) Fluorescence micrographs of <i>Prom_flp-19::gfp</i> expression in a wild-type animal, an <i>ets-5</i> mutant and an <i>ets-5</i> mutant carrying a wild-type copy of the <i>ets-5</i> locus in a fosmid-derived transgene. BAGL/R neuron positions are marked by red circles and cells previously identified as AWAL/R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034014#pone.0034014-Kim1" target="_blank">[19]</a> are marked by blue circles. The nerve ring is indicated by an arrowhead. The scale bar in lower panel is 20 µm. A: anterior, L: left. The <i>ets-5</i> mutant allele was <i>tm1734</i>. The <i>Prom_flp-19::gfp</i> transgene was <i>ynIs34</i> and the <i>ets-5</i> rescuing transgene was <i>rpEx246</i>.</p

ETS-5 is expressed by a subset of head neurons, including the chemosensitive BAG neurons.

<p>Ventral view of an adult hermaphrodite expressing an <i>ets-5::gfp</i> translational reporter transgene. BAG neurons, which express a <i>Prom_flp-17::dsRed</i> reporter, are indicated with arrows. Amphid neurons likely to be AWA neurons are indicated by arrowheads. A: anterior, R: right, the scale bar in lower panel is 20 µm. The <i>ets-5::gfp</i> transgene is <i>wzIs80</i> and the <i>Prom_flp-17::dsRed</i> transgene is <i>wzEx36</i>.</p

ETS-5 directly interacts with the <i>gcy-9</i> promoter.

<p>(A) ETS-5::GFP associates with the <i>gcy-9</i> promoter <i>in vivo</i>. Anti-GFP immunoprecipitates were prepared from cross-linked extracts of wild-type animals or animals carrying a functional <i>ets-5::gfp</i> transgene and interrogated for the presence of <i>gcy-9</i> promoter sequences by PCR. Immunoprecipitates from transgenic animals were enriched for <i>gcy-9</i> promoter sequences that contained the ETS-binding site at −202 bp. Control sequences at −5000 bp were not enriched in immunoprecipitates from transgenic animals. The <i>ets-5::gfp</i> transgene used was <i>wzIs80</i>. (B) ETS-5 binds to <i>gcy-9</i> promoter sequences <i>in vitro</i>. A mobility shift assay was performed with recombinant GST::ETS-5 and a 45 bp biotinylated DNA duplex probe containing the ETS-binding site from the <i>gcy-9</i> promoter. Recombinant GST::ETS-5 but not GST alone altered the electrophoretic mobility of the probe. The interaction between GST::ETS-5 and the probe was blocked by a molar excess of unlabeled probe but not by an excess of scrambled probe with the same nucleotide composition. Excess unlabeled wild-type and scrambled competitor probe was added in the following molar ratios: 10×, 50×, 100×, 500×.</p

The BAG neurons of <i>ets-5</i> mutants are defective in sensory transduction.

<p>(A) <i>ets-5</i> mutants are defective in a BAG-neuron-dependent CO₂ avoidance behavior. Plotted are the mean fractions of animals ± SEM that reversed during a four second exposure to either control atmosphere (0% CO₂, 20% O₂, balance N₂) or CO₂-enriched atmosphere (10% CO₂, 20% O₂, balance N₂). Strains tested were: the wild-type strain N2, the BAG-ablated strain CX11697, the <i>ets-5</i> mutant strain FX1734, which carries the <i>tm1734</i> deletion allele of <i>ets-5</i>, and a derivative of FX1734 that carries the <i>ets-5::gfp</i> transgene <i>wzIs80</i>. <i>N</i> = 3–5 populations of 30–50 animals. (B) The effect of <i>ets-5</i> mutation on CO₂ avoidance behavior is comparable to that of BAG neuron ablation. An avoidance index was calculated by subtracting the fraction of animals in a population that reversed in response to exposure to control atmosphere from the fraction that reversed in response to CO₂-enriched atmosphere. Plotted are the mean avoidance indices for each of the four strains tested ± SEM. P values were calculated by one-way ANOVA. <i>N</i> = 3–5 populations of 30–50 animals. (C) Wild-type BAG neurons show robust calcium responses to a CO₂ stimulus. Wild-type animals carrying a <i>Prom_gcy-9::cameleon</i> transgene, which drives expression of cameleon specifically in BAG neurons, were immobilized and exposed to a 10 s pulse of 10% CO₂. Plotted is the mean fractional ratio change in YFP/CFP emissions. The shaded area represents S.E.M. The cameleon expression transgene used was <i>wzIs82.</i> (D) The BAG neurons of <i>ets-5</i> mutants show reduced calcium responses to a CO₂ stimulus. Animals carrying a variant <i>Prom_gcy-33::cameleon</i> transgene, which drives <i>ets-5-</i>independent expression of cameleon in BAG neurons, were immobilized and exposed to a 10 s pulse of 10% CO₂. Plotted is the mean fractional ratio change in YFP/CFP emissions. The shaded area represents S.E.M. The cameleon expression transgene used was <i>wzEx56</i>.</p

ETS-binding sites in the <i>gcy-9</i> promoter are required for its BAG-neuron expression.

<p>(A) Fluorescence micrographs of <i>Prom_gcy-9::gfp</i> expression in a wild-type animal and an <i>ets-5</i> mutant. <i>Prom_gcy-9::gfp</i> expression was principally in BAG neurons (top panel) and was lost in the <i>ets-5</i> mutant (bottom panel). In the lower panel, the BAGL neuron position is marked by a red circle. The scale bar in lower panel is 20 µm. A: anterior, V: ventral. The <i>ets-5</i> mutant allele was <i>tm1734</i>. The <i>Prom_gcy-9::gfp</i> transgene was <i>wzEx37</i>. (B) The ETS domain of ETS-5 is most similar to that of mammalian Pet1. The ETS domains of ETS-5 and 26 mammalian ETS-family transcription factors were identified using the SMART database. Sequences were aligned using the CLUSTALW algorithm and sequence distances were plotted as a dendrogram using the Phylip sequence analysis package. (C) The <i>gcy-9</i> promoter contains an evolutionarily conserved ETS-binding motif that is highly similar to the Pet1-binding consensus. A logo of the Pet1-binding consensus sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034014#pone.0034014-Wei1" target="_blank">[24]</a> is shown aligned to sequences approximately 200 bp upstream of <i>gcy-9</i> coding sequences from <i>C. elegans</i> and three other rhabditid nematodes. (D) ETS binding motifs in the <i>gcy-9</i> promoter are required for promoter function. Shown are ventral views of adult hermaphrodites carrying a 1.9 kb fragment of the <i>gcy-9</i> promoter fused to <i>gfp</i> coding sequences, or a promoter variant that either carries an 88 bp deletion centered around the ETS binding site at −202 bp, or a variant that carries four-base substitutions in the ETS-binding sites. In each case two lines were tested for GFP expression. Red circles indicate the location of BAG neurons in the animal carrying the mutant transgene. The wild-type <i>gcy-9</i> promoter transgene used was <i>wzEx37</i> and the mutant promoter transgene was <i>wzEx38</i>.</p

Nusinersen versus Sham Control in Later-Onset Spinal Muscular Atrophy

International audienceBACKGROUND Nusinersen is an antisense oligonucleotide drug that modulates pre-messenger RNA splicing of the survival motor neuron 2 (SMN2) gene. It has been developed for the treatment of spinal muscular atrophy (SMA). METHODS We conducted a multicenter, double-blind, sham-controlled, phase 3 trial of nusinersen in 126 children with SMA who had symptom onset after 6 months of age. The children were randomly assigned, in a 2: 1 ratio, to undergo intrathecal administration of nusinersen at a dose of 12 mg (nusinersen group) or a sham procedure (control group) on days 1, 29, 85, and 274. The primary end point was the least-squares mean change from baseline in the Hammersmith Functional Motor Scale-Expanded (HFMSE) score at 15 months of treatment; HFMSE scores range from 0 to 66, with higher scores indicating better motor function. Secondary end points included the percentage of children with a clinically meaningful increase from baseline in the HFMSE score (>= 3 points), an outcome that indicates improvement in at least two motor skills. RESULTS In the prespecified interim analysis, there was a least-squares mean increase from baseline to month 15 in the HFMSE score in the nusinersen group (by 4.0 points) and a least-squares mean decrease in the control group (by -1.9 points), with a significant between-group difference favoring nusinersen (least-squares mean difference in change, 5.9 points; 95% confidence interval, 3.7 to 8.1; P< 0.001). This result prompted early termination of the trial. Results of the final analysis were consistent with results of the interim analysis. In the final analysis, 57% of the children in the nusinersen group as compared with 26% in the control group had an increase from baseline to month 15 in the HFMSE score of at least 3 points (P< 0.001), and the overall incidence of adverse events was similar in the nusinersen group and the control group (93% and 100%, respectively). CONCLUSIONS Among children with later-onset SMA, those who received nusinersen had significant and clinically meaningful improvement in motor function as compared with those in the control group. (Funded by Biogen and Ionis Pharmaceuticals; CHERISH ClinicalTrials. gov number, NCT02292537.