Dissecting the Serotonergic Food Signal Stimulating Sensory-Mediated Aversive Behavior in C. elegans

Nutritional state often modulates olfaction and in Caenorhabditis elegans food stimulates aversive responses mediated by the nociceptive ASH sensory neurons. In the present study, we have characterized the role of key serotonergic neurons that differentially modulate aversive behavior in response to changing nutritional status. The serotonergic NSM and ADF neurons play antagonistic roles in food stimulation. NSM 5-HT activates SER-5 on the ASHs and SER-1 on the RIA interneurons and stimulates aversive responses, suggesting that food-dependent serotonergic stimulation involves local changes in 5-HT levels mediated by extrasynaptic 5-HT receptors. In contrast, ADF 5-HT activates SER-1 on the octopaminergic RIC interneurons to inhibit food–stimulation, suggesting neuron-specific stimulatory and inhibitory roles for SER-1 signaling. Both the NSMs and ADFs express INS-1, an insulin-like peptide, that appears to cell autonomously inhibit serotonergic signaling. Food also modulates directional decisions after reversal is complete, through the same serotonergic neurons and receptors involved in the initiation of reversal, and the decision to continue forward or change direction after reversal is dictated entirely by nutritional state. These results highlight the complexity of the “food signal” and serotonergic signaling in the modulation of sensory-mediated aversive behaviors

RHGF-2 Is an Essential Rho-1 Specific RhoGEF that binds to the Multi-PDZ Domain Scaffold Protein MPZ-1 in Caenorhabditis elegans

RhoGEF proteins activate the Rho family of small GTPases and thus play a key role in regulating fundamental cellular processes such as cell morphology and polarity, cell cycle progression and gene transcription. We identified a Caenorhabditis elegans RhoGEF protein, RHGF-2, as a binding partner of the C. elegans multi-PDZ domain scaffold protein MPZ-1 (MUPP1 in mammals). RHGF-2 exhibits significant identity to the mammalian RhoGEFs PLEKHG5/Tech/Syx and contains a class I C-terminal PDZ binding motif (SDV) that interacts most strongly to MPZ-1 PDZ domain eight. RHGF-2 RhoGEF activity is specific to the C. elegans RhoA homolog RHO-1 as determined by direct binding, GDP/GTP exchange and serum response element-driven reporter activity. rhgf-2 is an essential gene since rhgf-2 deletion mutants do not elongate during embryogenesis and hatch as short immobile animals that arrest development. Interestingly, the expression of a functional rhgf-2::gfp transgene appears to be exclusively neuronal and rhgf-2 overexpression results in loopy movement with exaggerated body bends. Transient expression of RHGF-2 in N1E-115 neuroblastoma cells prevents neurite outgrowth similar to constitutive RhoA activation in these cells. Together, these observations indicate neuronally expressed RHGF-2 is an essential RHO-1 specific RhoGEF that binds most strongly to MPZ-1 PDZ domain eight and is required for wild-type C. elegans morphology and growth

Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from the insect trypanosomatid, Crithidia fasciculata, a member of the most primitive eukaryotic group to contain mitochondria. Separation of the purified PDC by SDS-PAGE yielded five bands of 70 (p70), 60 (p60), 55, 46 and 36.5 kDa, which appeared to correspond to dihydrolipoyl dehydrogenase binding protein (E3BP), dihydrolipoyl transacetylase (E2), E3, E1α and E1β, respectively. The purified complex did not exhibit endogenous PDH a kinase activity. p70 was much less abundant than p60. Polyclonal antisera raised against p70 did not cross-react with p60, and antisera raised against p60 did not cross-react with p70, suggesting that p60 did not arise from p70 by proteolysis. Both p70 and p60 contained similar amino terminal sequences. Both sequences contained the MPALSP motif similar to sequences present in both E3BP and E2 from other sources. Incubation of the purified PDC with [2- 14C]pyruvate in the absence of CoA resulted in the acetylation of both p70 and p60, suggesting that both proteins contained lipoyl domains, but the specific incorporation of label into p70 was significantly greater than for p60. Limited proteolysis of the acetylated complex with trypsin yielded two major fragments derived from p60 of 35 and 30 kDa, corresponding to E2 l and E2 i , and one major acetylated fragment of 58 kDa derived from p70. Therefore, these results suggest that p70 is an E3BP and given its apparent M r and degree of acetylation, it contains multiple lipoyl domains

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets

Dual Excitatory and Inhibitory Serotonergic Inputs Modulate Egg Laying in Caenorhabditis elegans

Serotonin (5-HT) regulates key processes in both vertebrates and invertebrates. Previously, four 5-HT receptors that contributed to the 5-HT modulation of egg laying were identified in Caenorhabditis elegans. Therefore, to assess potential receptor interactions, we generated animals containing combinations of null alleles for each receptor, especially animals expressing only individual 5-HT receptors. 5-HT-stimulated egg laying and egg retention correlated well with different combinations of predicted excitatory and inhibitory serotonergic inputs. For example, 5-HT did not stimulate egg laying in ser-1, ser-7, or ser-7 ser-1 null animals, and ser-7 ser-1 animals retained more eggs than wild-type animals. In contrast, 5-HT-stimulated egg laying in ser-4;mod-1 animals was greater than in wild-type animals, and ser-4;mod-1 animals retained fewer eggs than wild-type animals. Surprisingly, ser-4;mod-1;ser-7 ser-1 animals retained the same number of eggs as wild-type animals and exhibited significant 5-HT-stimulated egg laying that was dependent on a previously uncharacterized receptor, SER-5. 5-HT-stimulated egg laying was absent in ser-5;ser-4;mod-1;ser-7 ser-1 animals, and these animals retained more eggs than either wild-type or ser-4;mod-1;ser-7 ser-1 animals. The 5-HT sensitivity of egg laying could be restored by ser-5 muscle expression. Together, these results highlight the dual excitatory/inhibitory serotonergic inputs that combine to modulate egg laying

PAPP paralyzes <i>C</i>. <i>elegans</i> via SER-4 and DOP-3.

<p><b>A-C.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic non-NGM agar plates. <b>A.</b> PAPP (0.5 mM)-dependent paralysis of wild-type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>B.</b> Dose-response curves for PAPP-dependent paralysis at 15 min exposure for wild type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). <b>C.</b> PAPP (0.5 mM)-dependent paralysis of 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing P<i>dop-3</i>::<i>dop-3</i> RNAi. Data are presented as mean ± SE (n = 3). ‘*’ p≤0.001, significantly different from 5-HT <i>quint</i> animals assayed under identical conditions.</p