Neuropeptide inactivation regulates egg-laying behavior to influence reproductive health in Caenorhabditis elegans

Abstract

Neural communication requires both fast-acting neurotransmitters and neuromodulators that function on slower timescales to communicate. Endogenous bioactive peptides, often called "neuropeptides," comprise the largest and most diverse class of neuromodulators that mediate crosstalk between the brain and peripheral tissues to regulate physiology and behaviors conserved across the animal kingdom. Neuropeptide signaling can be terminated through receptor binding and internalization or degradation by extracellular enzymes called neuropeptidases. Inactivation by neuropeptidases can shape the dynamics of signaling in vivo by specifying both the duration of signaling and the anatomic path neuropeptides can travel before they are degraded. For most neuropeptides, the identity of the relevant inactivating peptidase(s) is unknown. Here, we established a screening platform in C. elegans utilizing mass spectrometry-based peptidomics to discover neuropeptidases and simultaneously profile the in vivo specificity of these enzymes against each of more than 250 endogenous peptides. We identified NEP-2, a worm ortholog of the mammalian peptidase neprilysin-2, and demonstrated that it regulates specific neuropeptides, including those in the egg-laying circuit. We found that NEP-2 is required in muscle cells to regulate signals from neurons to modulate both behavior and health in the reproductive system. Taken together, our results demonstrate that peptidases, which are an important node of regulation in neuropeptide signaling, affect the dynamics of signaling to impact behavior, physiology, and aging.

Keywords: C. elegans; aging; behavior; egg laying; healthspan; matricide; neural circuit; neuropeptidase; neuropeptide; reproductive aging.

Figure 1:. NEP-2 is a neuropeptidase that regulates levels of NLP-3 neuropeptides.

(A) Schematic of nep-2 gene structure with annotations of the alleles used in this study, ok2846 is the primary allele utilized. (B) Schematic of the workflow for label-free mass spectrometry. (C) (top) Schematic of steps for neuropeptide processing from a neuropeptide proprotein precursor. (bottom) Schematic of NLP-3 proprotein sequence with individual peptides and processing enzyme cleavage sites annotated. (D-H) Relative quantification of each NLP-3 neuropeptide. Peak area for each replicate is plotted and compared between the two strains, wild-type and nep-2(lof), for each neuropeptide. Table of calculated fold change noted in each graph below neuropeptide name. (n = 9–10 biological replicates of 50,000 worms) Error bars indicate mean ± s.e.m. Statistical tests: t-test. p-values indicated by * p < 0.05, ^** p < 0.01. See also Figure S1.

Figure 2:. Loss of nep-2 causes hyperactive egg-laying behavior.

(A) Diagram of the egg-laying circuit that results in egg-release by the animal^,,,– and proposed hypothesis for observations in nep-2(lof). The command neuron, HSN, releases serotonin and NLP-3 neuropeptides to promote egg-laying behavior. Egg-laying is coordinated by the vulval (vm) and uterine (um) muscles. After egg-release, uv1 cells release tyramine and neuropeptides to inhibit HSN activity. If there is increase in NLP-3 neuropeptide abundance in nep-2(lof) animals, this would result in hyperactive egg-laying. (B) Total number of eggs in the uterus of day 2 adult wild-type N2 animals and day 2 adult nep-2(lof) animals. (n = 45, 47 animals) (C) Proportion of eggs in the uterus that are at or below 8-cell stage versus greater than 8-cell stage in day 2 adult animals. (n = 45, 47 animals) (D) Stage of eggs laid by day 2 adult animals. (n = 83, 110 eggs) (E) Total number of eggs in the uterus of day 2 adult wild-type N2 animals, day 2 adult nep-2(lof) animals, and day 2 adult nep-2(lof) expressing a rescue construct. (n = 18, 24, 19 animals) (F) Proportion of eggs in the uterus that are at or below 8-cell stage versus greater than 8-cell stage in day 2 adult animals. (n = 18, 24, 19 animals) G) Stage of eggs laid by day 2 adult animals. (n = 101, 131, 146 eggs) (H) Total number of eggs in the uterus of day 2 adult wild-type N2 animals, day 2 adult nep-2(lof) animals, and day 2 adult wild-type N2 animals expressing extra copies of nep-2. (n = 36, 36, 22 animals) (I) Proportion of eggs in the uterus that are at or below 8-cell stage versus greater than 8-cell stage in day 2 adult animals. (n = 36, 36, 22 animals) (J) Stage of eggs laid by day 2 adult animals. (n = 98, 64, 96 eggs) Eggs are normally laid after the 26-cell stage, then develop outside the uterus through three stages (comma, plum, pretzel) during which the embryo adopts a worm-like shape, ultimately leading to hatching. (K) Percent relative frequency of number of eggs per lay. (n = 224, 113 eggs) Error bars indicate mean ± s.e.m. Statistical tests: t-test for (B), 2-way ANOVA for (C, F, I), one-way ANOVA for (E, H), Fisher's exact test for (D, G, J, K). p-values indicated by ^** p < 0.01, ^**** p < 0.0001. See also Figure S2.

Figure 3:. Hyperactive egg-laying in nep-2(lof) animals results from dysregulated NLP-3 signaling.

(A) Total number of eggs in the uterus of day 2 adult wild-type N2 animals, day 2 adult nep-2(lof) animals, day 2 adult nlp-3(lof) animals, and day 2 nep-2; nlp-3 animals. (n = 47, 48, 35, 35 animals) (B) Proportion of eggs in the uterus that are at or below 8-cell stage versus greater than 8-cell stage in day 2 adult animals. (n = 47, 48, 35, 35 animals) (C) Stage of eggs laid by day 2 adult animals. (n = 175, 157, 98, 167 eggs) Error bars indicate mean ± s.e.m. Statistical tests: one-way ANOVA for (A), 2-way ANOVA for (B), Fisher's exact test for (C). p-values indicated by ^** p < 0.01, ^**** p < 0.0001. See also Figure S3.

Figure 4:. NEP-2 activity in muscle regulates the peptidergic arm of the egg-laying circuit.

(A) Fluorescent images (left) of wild-type animals expressing endogenous knock-in reporter for NEP-2 (nep-2p::GFPnovo2::nep-2) indicating expression in uterine muscles, vulval muscles, and body wall muscle. Schematic of muscles in the egg-laying apparatus (right). (B) Dot plot of the proportion and relative expression of nep-2 in different tissues from Calico sc-RNA-seq dataset. (C) Relative expression of nep-2 measured by qPCR through adulthood lifespan of wild-type N2 animals. Relative expression normalized to day 1 animals. (n = 3 biological replicates) (D) Stage of eggs laid by wild-type day 2 adult animals with either control RNAi or RNAi targeting nep-2. (n = 109, 172 eggs) (E) Stage of eggs laid by day 2 adult animals that are RNAi-competent only in neurons with either control RNAi or RNAi targeting nep-2. (n = 106, 124 eggs) (F) Stage of eggs laid by day 2 adult wild-type N2 animals, day 2 adult nep-2(lof) animals, and day 2 adult nep-2(lof) expressing a rescue construct only in muscle cells. (n = 125, 54, 97 eggs) (G) Dot plot of the proportion and relative expression of nep-2 in each cell type of the egg-laying circuit from Calico sc-RNA-seq dataset. (H) Percent relative frequency of number of eggs per lay. (n = 224, 113, 171 eggs) (I) Total number of eggs in the uterus of day 2 adult wild-type N2 animals and day 2 adult nep-2(lof) animals, both with control RNAi and RNAi targeting npr-36. (n = 39, 41, 40, 41 animals) (J) Proportion of eggs in the uterus that are at or below 8-cell stage versus greater than 8-cell stage in day 2 adult animals. (n = 39, 41, 40, 41 animals) (K) Stage of eggs laid by day 2 adult animals. Eggs are laid after the 26-cell stage by wild-type animals, and comma is an embryo development stage that normally occurs outside of the uterus. (n = 103, 158, 102, 107 eggs) (L) Dot plot of the proportion and relative expression of npr-36 in each cell type of the egg-laying circuit from Calico sc-RNA-seq dataset. (M) Proposed model of NEP-2 function in the egg-laying circuit to regulate NLP-3 neuropeptide levels and consequently, egg-release. NEP-2 is a neuropeptidase expressed in muscle cells (vulval and uterine) that regulates NLP-3 neuropeptides released from HSN neurons in order to maintain uterine quiescence and prevent aberrant egg-laying behavior. Error bars indicate mean ± s.e.m. Statistical tests: Fisher's exact test for (D-F, H, K), one-way ANOVA for (I), 2-way ANOVA for (J). p-values indicated by * p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. See also Figure S4.

Figure 5:. NEP-2 impacts matricide and improves healthspan and lifespan by modulating the egg-laying circuit.

(A) Cumulative percentage of animals experiencing either bagging or bursting of vulva through the reproductive span. (n = 120 animals per strain) (B) Total number of eggs in the uterus of day 2 adult wild-type N2 animals, day 2 adult nep-2(lof) animals, day 2 adult daf-2(lof) animals, and day 2 adult nep-2; daf-2 animals. (n = 24, 30, 33, 35 animals) (C) Proportion of eggs in the uterus that are at or below 8-cell stage versus greater than 8-cell stage in day 2 adult animals. (n = 24, 30, 33, 35 animals) (D) Total number of eggs in the uterus of day 4 adult wild-type N2 animals, day 4 adult nep-2(lof) animals, day 4 adult daf-2(lof) animals, and day 4 adult nep-2; daf-2 animals. (n = 31, 30, 29, 33 animals) (E) Proportion of eggs in the uterus that are at or below 8-cell stage versus greater than 8-cell stage in day 2 adult animals. (n = 31, 30, 29, 33 animals) (F) Lifespan analysis. (n = 120 animals per strain) (G) Lifespan analysis of daf-2(lof) and nep-2; daf-2 with (filled circles) and without (open circles) FUDR. (n = 120 animals per strain) (H) Survival curve of animals chronically exposed to 10 mM paraquat starting at day 1 of adulthood. (n = 100, 100, 150, 100 animals) (I) Lifespan analysis of animals mated for 24 hours from L4. (n = 22, 20 animals) (J) Cumulative percentage of animals experiencing bagging through the reproductive span. (n = 22, 20 animals) Error bars indicate mean ± s.e.m. Statistical tests: one-way ANOVA for (B, D), 2-way ANOVA for (C, E). p-values indicated by * p < 0.05, ^*** p < 0.001, ^**** p < 0.0001. See also Figure S5, Table S2, and Video S1.