RIC-7 promotes neuropeptide secretion

Abstract

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV-mediated secretion.

Figure 1. Aldicarb response and locomotion defects in ric-7 mutants.

(A) A schematic illustrating the ric-7 gene structure is shown. Deleted regions (bar), point mutations (arrows), and the site utilized for GFP/mCherry tagging are indicated. (B–C) Aldicarb-induced paralysis (B) and locomotion rate (C) are compared for the indicated genotypes. The number of trials (∼20 animals/trial, B) or the number of animals analyzed (C) are indicated for each genotype. For rescue experiments, ric-7 transgenes are indicated as follows: ACh (unc-17 promoter), GABA (unc-25 promoter), pan-neuron (snb-1 promoter), muscle (myo-3 promoter). Values that differ significantly from wild type (**, p<0.001, Student's t-test) and from ric-7 mutants (#, p<0.01; ##, p<0.001, Student's t-test) are indicated. Error bars indicate SEM.

Figure 2. Expression pattern of RIC-7.

(A) The ric-7 promoter expresses nuclear localized Cherry (HIS-24::wCherry) primarily in the nervous system of an adult worm. Anterior is left; ventral is up. The asterisk indicates fluorescence encoded by a co-injection marker. (B) The ric-7 promoter is expressed in cholinergic (top panel) and GABAergic (bottom panel) motor neurons in the ventral cord. Cell bodies of cholinergic (unc-17 promoter) and GABAergic (unc-30 promoter) neurons were identified by expression of the indicated GFP reporter constructs. (C) Distribution of RIC-7, a synaptic vesicle marker (UNC-57 Endophilin) (top panel), and a DCV marker (NLP-21) (bottom panel) are compared in the dorsal cord axons of cholinergic motor neurons. (D) Distribution of RIC-7::GFP in cholinergic motor neurons of unc-104 KIF1A mutants. Cell bodies (arrow) and ventral cord processes (arrow heads) are indicated.

Figure 3. Body muscle responses to ACh and GABA are unaltered in ric-7 mutants.

(A) Time course of levamisole (200 µM) induced paralysis is shown for wild type and ric-7(nu447) adults. Three trials (∼20 animals/trial) were performed for each genotype. (B) ACh (top) and Muscimol (bottom)-activated currents were recorded from body wall muscles of ric-7 and wild type adults. Representative responses (left) and summary data (right) are shown. Wild type and ric-7 mutant responses were not significantly different. (C–D) Representative images (above) and summary data (below) are shown for ACR-16::GFP (C) and UNC-49::GFP (D) expressed in body muscles of wild type and ric-7 adults (using the myo-3 promoter). No significant differences were observed. The number of animals analyzed is indicated for each genotype (panels B–D). Error bars indicate SEM.

Figure 4. Baseline ACh release is unaltered in ric-7 mutants.

Endogenous EPSCs (A) and stimulus-evoked EPSCs (B) were recorded from body wall muscles of wild type and ric-7(nu447) adults. Representative traces of endogenous EPSCs (A), averaged traces of stimulus-evoked responses (B), and summary data for both are shown. The number of animals analyzed is indicated for each genotype. No significant differences were observed. (C) Representative images (left) and summary data (right) are shown for GFP-tagged SNB-1 in dorsal cord axons of cholinergic motor neurons (expressed with the unc-129 promoter) in wild type and ric-7 adults. The number of animals analyzed is indicated for each genotype. No significant differences were observed. Error bars indicate SEM.

Figure 5. Neuropeptide processing mutations and

The paralytic response to aldicarb treatment was analyzed in strains containing mutations that inactivate pro-neuropeptide processing enzymes (egl-3 PC2 and egl-21 CPE), or those inactivating RIC-7. The number of trials (∼20 animals/trial) is shown for each genotype. Values that differ significantly from wild type (*, p<0.01; **, p<0.001, Students t-test) are indicated. Error bars indicate SEM. Values that are not significantly different are indicated (ns).

Figure 6. RIC-7 promotes neuropeptide release.

YFP-tagged NLP-21 and INS-22 were expressed in cholinergic motor neurons using the unc-129 promoter. Representative images (A) and summary data (B) are shown for NLP-21 (top) and INS-22 (bottom) fluorescence in dorsal cord axons of the indicated genotypes. The number of animals analyzed is indicated for each genotype. (C–D) Representative images (C) and summary data (D) are shown for NLP-21 and INS-22 fluorescence in coelomocytes of the indicated genotypes. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type (**, p<0.001, ***, p<0.0001 Students t-test) and from ric-7 mutants (#, p<0.01, ##, p<0.001, Students t-test) are indicated. Error bars indicate SEM. For rescue experiments, ric-7 transgenes are as follows: pan-neuron (snb-1 promoter), DA neuron (unc-129 promoter).

Figure 7. NLP-12 secretion is decreased in ric-7 mutants.

(A) YFP-tagged NLP-12 was expressed in DVA neurons (using the nlp-12 promoter). Representative images (A) and summary data (B) are shown for NLP-12 puncta fluorescence in the indicated genotypes. The number of animals analyzed is indicated for each genotype. (C) Aldicarb-induced paralysis was quantified for the indicated genotypes. The number of trials (∼20 animals/trial) is indicated for each genotype. (D–E) Stimulus evoked EPSCs were recorded from adult body wall muscles of the indicated genotypes. Recordings were done after a 60 minute aldicarb pre-treatment (gray) or in untreated controls (black). Averaged evoked responses (D) and summary data (E) are shown. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type (**, p<0.001, ***, p<0.0001, Students t-test) and from ric-7 mutants (##, p<0.001, ###, p<0.0001 Students t-test) are indicated. Error bars indicate SEM. For rescue experiments, ric-7 transgenes are as follows: DVA (nlp-12 promoter).

Figure 8. Analysis of GABA transmission in ric-7 mutants.

(A) Intestinal muscle contractions during the defecation motor program (quantified as expulsions/pBoc) were analyzed in the indicated genotypes. (B) Endogenous IPSCs were recorded from adult body wall muscles of the indicated genotypes. Representative traces (left), and summary data (right) are shown. The number of animals analyzed is indicated for each genotype. (C) Representative images (left) and summary data (right) for GFP::SNB-1 (expressed by the unc-25 promoter) in dorsal cord axons of the indicated genotypes. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type (**, p<0.001, ***, p<0.0001 Students t-test) and from ric-7 mutants (#, p<0.05, ##, p<0.001, ###, p<0.0001 Students t-test) are indicated. Error bars indicate SEM. For rescue experiments, ric-7 transgenes are as follows: ACh (unc-17 promoter), GABA (unc-47 promoter), pan-neuron (snb-1 promoter), intestine (vha-6 promoter).