CCHamide-2 Is an Orexigenic Brain-Gut Peptide in <i>Drosophila</i>

<div><p>The neuroendocrine peptides CCHamide-1 and -2, encoded by the genes <i>ccha1</i> and <i>-2</i>, are produced by endocrine cells in the midgut and by neurons in the brain of <i>Drosophila melanogaster</i>. Here, we used the CRISPR/Cas9 technique to disrupt the <i>ccha1</i> and <i>-2</i> genes and identify mutant phenotypes with a focus on <i>ccha-2</i> mutants. We found that both larval and adult <i>ccha2</i> mutants showed a significantly reduced food intake as measured in adult flies by the Capillary Feeding (CAFE) assay (up to 72% reduced food intake compared to wild-type). Locomotion tests in adult flies showed that <i>ccha2</i> mutants had a significantly reduced locomotor activity especially around 8 a.m. and 8 p.m., where adult <i>Drosophila</i> normally feeds (up to 70% reduced locomotor activity compared to wild-type). Reduced larval feeding is normally coupled to a delayed larval development, a process that is mediated by insulin. Accordingly, we found that the <i>ccha2</i> mutants had a remarkably delayed development, showing pupariation 70 hours after the pupariation time point of the wild-type. In contrast, the <i>ccha-1</i> mutants were not developmentally delayed. We also found that the <i>ccha2</i> mutants had up to 80% reduced mRNA concentrations coding for the <i>Drosophila</i> insulin-like-peptides-2 and -3, while these concentrations were unchanged for the <i>ccha1</i> mutants. From these experiments we conclude that CCHamide-2 is an orexigenic peptide and an important factor for controlling developmental timing in <i>Drosophila</i>.</p></div

Nucleotide sequences and corresponding amino acid sequences around the deletions in two <i>ccha1</i> (A) and two <i>ccha2</i> (B) mutants.

<p>In the wild-type these nucleotide sequences code for the unprocessed CCHamide peptides, which are shown in red at the top of each panel. The black arrows in these red lines at the top indicate the initial cleavage steps in each prohormone, catalyzed by prohormone convertase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.ref038" target="_blank">38</a>]. A. Parts of the DNA sequences from the two <i>ccha1</i> mutants (<i>ccha1</i>^<i>SK4</i> and <i>ccha1</i>^<i>SK8</i>) and the corresponding wild-type DNA sequence coding for CCHamide-1. Mutant <i>ccha1</i>^<i>SK4</i> lacks 5 base pairs (bp), while mutant <i>ccha</i>^<i>SK8</i> lacks 13 bp. Both deletions lead to a frameshift, so that no intact CCHamide-1 peptide can be produced. For example, while in the wild-type the two cysteine residues (underlined) form a cystine bridge, such ring structure can not be formed in the mutant peptides, because a second cysteine residue is lacking. Furthermore, while in the wild-type processing occurs between the KR and S amino acid sequence (arrow), followed by a conversion of the C-terminal G residue into a C-terminal amide [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.ref038" target="_blank">38</a>], such posttranslational processings can not occur in the two mutants, due to the lack of the GKR amino acid sequence at these positions. The mutations, therefore, result in nonfunctional peptides that only have the N-terminal amino acid residues in common with wild-type CCHamide-1. B. Parts of the DNA sequences from <i>ccha2</i> mutants and their corresponding wild-type DNA sequences coding for CCHamide-2. The two mutants have identical 10 bp deletions that, again, cause a frameshift in the reading frame, resulting in the loss of the cystine bridge and the appropriate processing sites to yield functional peptides. Furthermore, the two mutants have a premature stop codon (TGA).</p

qPCR of <i>Drosophila</i> insulin-like peptide (DILP) gene expressions in third instar larvae and pupae of ccha1 and -2 null mutants and wild-type animals.

<p>Control animals are indicated by black bars, <i>ccha1</i> mutants are indicated by white bars, <i>ccha2</i> mutants are indicated by red bars. The vertical bars represent S.E.M. (n = 3). Thirty animals were used in each measurement (2 technical replicates; 3 biological replicates). A. In larval <i>ccha2</i> mutants, <i>dilp2</i> gene expression is reduced by about 50% (t-test, *** p≤0.001), while in larval <i>ccha1</i> mutants there is no reduction compared to wild-type. B. In pupal <i>ccha2</i> mutants (pupal stage P-5), <i>dilp2</i> gene expression is reduced to 35% of the wild-type values (t-test, *** p≤0.001), while there is no reduction in <i>ccha1</i> mutants. C. In larval <i>ccha2</i> mutants, <i>dilp3</i> gene expression is reduced to 20% of the wild-type values (t-test, *** p≤0.001), while there is no such downregulation in <i>ccha1</i> mutants. D. In pupal <i>ccha2</i> mutants (stage P-5), the <i>dilp3</i> gene expression is downregulated to about 50% of the wildtype values (t-test, *** p≤0.001), while there is no significant downregulation in the pupal <i>ccha1</i> mutants.</p

Pupariation time points of <i>ccha2</i> mutants compared to control.

<p>The horizontal line parallel to the abscissa indicates 50% of the animals having undergone pupariation. The vertical stippled lines indicate the time points, where 50% of the experimental animals have pupariated. Control animals (indicated by a black line) pupariated (pupal stage P-2) at 132 hrs after egg laying Homozygous mutants (indicated by a red line) pupariated at 202 hrs after egg laying and were, therefore, 70 hrs delayed compared to controls. Furthermore, <i>ccha2</i> mutants rescued by re-introducing the <i>ccha2</i> gene (indicated by a green line) pupariated at 148 hrs and were, thus rescued by 80%. The data points represent the average of five independent experiments, containing 15–25 animals each. The vertical bars represent S.E.M. The differences between control and <i>ccha2</i> mutants, and between <i>ccha2</i> mutants and rescued mutants are stastically significant (one-way ANOVA test, p≤0.001).</p

Expresion of the <i>ccha2</i> gene in different organs of mid third instar <i>D</i>. <i>melanogaster</i> larvae (92 hrs after egg laying).

<p>Two primer sets were used for qPCR: One primer set previously applied by us [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.ref013" target="_blank">13</a>] and one primer set used by Sano et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.ref018" target="_blank">18</a>] (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.s003" target="_blank">S1 Table</a>). (A) qPCR results using the primer set described by us [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.ref013" target="_blank">13</a>]. (B) qPCR results using the primer set described by Sano et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.ref018" target="_blank">18</a>]. It is clear from both experiments that the gut is the major source of <i>ccha2</i> mRNA, while the fat body is virtually devoid of <i>ccha2</i> mRNA (n = 3; student t-test *** p≤0.001).</p

Circadian activities of the control (black lines) and CCHamide-2 mutants (red lines).

<p>The activities were measured using a <i>Drosophila</i> activity monitor that monitors one-dimensional locomotion of single flies. Light is switched on at 8 a.m. and switched off at 8 p.m. The upper panel gives the activities of 6-d old male, and the lower panel of 6-d old female flies. The data points represent the average of three independent experiments containing 32 flies each (n = 3). The vertical bars represent S.E.M. When no vertical bars are visible, they are smaller than the symbols used. The green areas highlight time periods around 8 a.m. and 8 p.m., where the activity differences between mutants and wild-type were especially significant. These periods coincide with the normal feeding periods of wild-type <i>Drosophila</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.ref025" target="_blank">25</a>]. The arrows indicate significant activity differences between mutants and controls at 8 a.m. and 8 p.m. P-values are between p≤0.001 and p≤0.05.</p

Fecal Microbiota Transplantation in Systemic Sclerosis: A Double-Blind, Placebo-Controlled Randomized Pilot Trial

Objectives Systemic sclerosis (SSc) is an auto-immune, multi organ disease marked by severe gastrointestinal (GI) involvement and gut dysbiosis. Here, we aimed to determine the safety and efficacy of fecal microbiota transplantation (FMT) using commercially-available anaerobic cultivated human intestinal microbiota (ACHIM) in SSc. Methods Ten patients with SSc were randomized to ACHIM (n = 5) or placebo (n = 5) in a double-blind, placebo-controlled 16-week pilot. All patients had mild to severe upper and lower GI symptoms including diarrhea, distention/bloating and/or fecal incontinence at baseline. Gastroduodenoscopy transfer of ACHIM or placebo was performed at weeks 0 and 2. Primary endpoints were safety and clinical efficacy on GI symptoms assessed at weeks 4 and 16. Secondary endpoints included changes in relative abundance of total, immunoglobulin (Ig) A- and IgM-coated fecal bacteria measured by 16s rRNA sequencing. Results ACHIM side effects were mild and transient. Two placebo controls experienced procedure-related serious adverse events; one developed laryngospasms at week 0 gastroduodenoscopy necessitating study exclusion whilst one encountered duodenal perforation during gastroduodenoscopy at the last study visit (week 16). Decreased bloating, diarrhea and/or fecal incontinence was observed in four of five patients in the FMT group (week 4 or/and 16) and in two of four in the placebo group (week 4 or 16). Relative abundance, richness and diversity of total and IgA-coated and IgM-coated bacteria fluctuated more after FMT, than after placebo. Conclusions FMT of commercially-available ACHIM is associated with gastroduodenoscopy complications but reduces lower GI symptoms by possibly altering the gut microbiota in patients with SSc

Larval feeding assays for control flies (black bars), <i>ccha2</i> null mutants (red bars) are rescued <i>ccha2</i> mutants (green bars).

<p>A. Larval feeding assay measuring the frequency of mouth hook contractions of third instar larvae feeding on agar covered with a 2% yeast solution. The <i>ccha2</i> null mutants have 63% of their feeding activity left compared to the controls (n = 5; t-test. *** p≤0.001). The rescued mutants restored their feeding activity to a level which is 86% of the control activity. The difference between <i>ccha2</i> null mutants and rescued mutants is significant (t-test, * p≤0.5). B. A different larval feeding assay, measuring the amount of ingested color-labelled yeast per hour. The <i>ccha2</i> null mutants have 43% of their feeding activities left compared to controls (n = 5; t-test, *** p≤0.001). The rescued mutants restored their feeding activity to 78% of the controls. The difference between <i>ccha2</i> null and rescued mutants is significant (t-test, ** p≤0.01).</p

Hypothetical model for the actions of CCHamide-2 in <i>D</i>. <i>melanogaster</i>.

<p>A. When the lumen of the midgut (lower part of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.g008" target="_blank">Fig 8A</a>) is devoid of nutrients, the CCHamide-2 containing endocrine cells of the gut wall (highlighted in green) signal this information to the brain by releasing CCHamide-2 into the circulation (arrow 1). After binding to its brain receptors, CCHamide-2 induces foraging and feeding behavior. In addition to this long-distance CCHamide-2 signaling pathway, there is a short distance CCHamide-2 signaling pathway (arrow 2), where a small group of CCHamide-2 neurons in the brain (highlighted in green) also innervate the motor circuits underlying foraging and feeding. We hypothesize that these neurons might perhaps directly monitor the nutrients in the circulation. B. A flow diagram of the proposed sequence of events after CCHamide-2 has induced feeding (step 1; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133017#pone.0133017.g008" target="_blank">Fig 8A</a>). Feeding induces the release of DILPs (Step 2; see refs 26, 31, 32). DILPs stimulate growth (step 3; see refs 26, 28), but also induce satiety (step 4; see ref 30, 31, 33). It is assumed that satiety blocks the release of CCHamide-2 and other orexigenic neuropeptides.</p