Serotonin deficiency from constitutive SKN-1 activation drives pathogen apathy

Abstract

When an organism encounters a pathogen, the host innate immune system activates to defend against pathogen colonization and toxic xenobiotics produced. C. elegans employ multiple defense systems to ensure survival when exposed to Pseudomonas aeruginosa including activation of the cytoprotective transcription factor SKN-1/NRF2. Although wildtype C. elegans quickly learn to avoid pathogens, here we describe a peculiar apathy-like behavior towards PA14 in animals with constitutive activation of SKN-1, whereby animals choose not to leave and continue to feed on the pathogen even when a non-pathogenic and healthspan-promoting food option is available. Although lacking the urgency to escape the infectious environment, animals with constitutive SKN-1 activity are not oblivious to the presence of the pathogen and display the typical pathogen-induced intestinal distension and eventual demise. SKN-1 activation, specifically in neurons and intestinal tissues, orchestrates a unique transcriptional program which leads to defects in serotonin signaling that is required from both neurons and non-neuronal tissues. Serotonin depletion from SKN-1 activation limits pathogen defenses capacity, drives the pathogen-associated apathy behaviors and induces a synthetic sensitivity to selective serotonin reuptake inhibitors. Taken together, our work reveals interesting insights into how animals perceive environmental pathogens and subsequently alter behavior and cellular programs to promote survival.

Fig. 1. Activation of SKN-1 drives pathogen apathy.

A Graphical representation of the variations in food choice assays performed. B, C Apathy displayed by skn-1gf worms in the (B) traditional choice assay or (C) forced choice assays. The pathogen leaving response of WT worms is absent in response to the non-pathogenic strain PA14∆gacA (D) and resembles the apathy behavior of skn-1gf animals (E). Both WT and skn-1gf mutant animals display apathy for PA14 following depletion of wdr-23 by RNAi (F). Each of the food choice assays comprised of N ≥ 3 (with ≥ 150 worms per biological replicate per strain/condition) and analyzed via two-way ANOVA test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). Individual “p” value numbers for the comparisons within each graph are placed above the respective comparison bars in the graph. Source data for all behavioral responses are provided as a Source Data file 1.

Fig. 2. Pathogen apathy requires neuronal SKN-1.

Chemotaxis towards (A) attractants [diacetyl (DA) and isoamyl alcohol (IAA)] are similar for both WT and skn-1gf worms. B Graphical representation of pathogen training and subsequent food choice assay. Differences in pathogen leaving response in (C) WT and (D) skn-1gf worms following pathogen training for 0, 2, and 4 hr of exposure. Specific expression of SKN-1gf isoform a (E) or isoform c (F) in ASI neurons does not elicit failed pathogen leaving behaviors as observed in the skn-1gf mutant (G) Pan-neuronal degradation of SKN-1gf restores pathogen leaving behavior in the skn-1gf mutant. Each of the chemotaxis assays comprised of N ≥ 3 (with ≥ 150 worms per biological replicate per strain/condition) and analyzed via one-way ANOVA test data represented as mean values +/− SEM for the. Each of the food choice assays comprised of N ≥ 3 (with ≥ 150 worms per biological replicate per strain/condition) and analyzed via two-way ANOVA test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). Individual “p” value numbers for the comparisons within each graph are placed above the respective comparison bars in the graph. Source data for all behavioral responses are provided as a Source Data file 1.

Fig. 3. Precocious activation of intestinal SKN-1 in responses to PA14.

A–D Intestinal accumulation and stabilization of SKN-1-gf-GFP and SKN-1wt-GFP upon PA14 exposure in comparison to OP50 (time point: 30 mins); the first two intestinal nuclei of each worm are outlined by white-dotted circles and the constitutive expression of SKN-1 in the ASI neurons is marked with white arrows. The scale bar represents 25 µm. Quantification of (E) percent of the population and (F) the intensity of SKN-1 nuclear localization. G Intestine-specific degradation of SKN-1gf does not restore pathogen-leaving behavior in skn-1gf mutant. H Co-expression of the a and c isoforms of SKN-1gf in the intestine does not elicit apathy behavior, as observed in the skn-1gf mutant, while (I) dual expression in both neurons and the intestine induces a modest apathy response that resembles skn-1gf mutants. For the population studies, a total of 60 (N = 3; n = 20) animals were counted per time point per condition. For quantification studies, a minimum of three animals per time point per condition was taken into consideration. Each of the nuclear accumulation time points comprised of N ≥ 3 (with n ≥ 3 per biological replicate per strain/condition) and analyzed via two-way ANOVA test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). Each of the food choice assay comprised of N ≥ 3 (with ≥ 150 worms per biological replicate per strain/condition) and analyzed via two-way ANOVA test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). Individual “p” value numbers for the comparisons within each graph is placed above the respective comparison bars in the graph. Source data for all nuclear activation responses are provided as a Source Data file 2. Source data for all behavioral responses are provided as a Source Data file 1.

Fig. 4. Context dependent transcriptional signature of PA14 exposure.

A Principal component analysis of the four sample groups (WT - OP50, WT - PA14, skn-1gf - OP50, and skn-1gf - PA14) indicates unique and context-dependent transcriptional responses. B Analyses of the various classes of genes with unique transcriptional responses revealed similarities upon pairwise comparisons and three-way comparisons, along with identifying 1348 genes that are differentially expressed across comparisons. C Genotype-specific responses to PA14 identified 363 genes with reversed directionality of transcriptional response between WT and skn-1gf animals (170 down-regulated and 193 up-regulated in WT in comparison to skn-1gf). 579 genes with similar directionality but the differential magnitude of the transcriptional response between WT and skn-1gf mutants (322 down-regulated and 257 up-regulated). RNAseq data is available at the NIH (GEO) Gene Expression Omnibus (GSE251677). Source data for all Genotype-specific transcriptional responses are provided as a Source Data file 3.

Fig. 5. Constitutive SKN-1 Activation results in serotonin limitations.

A Heat map of neuronal enrichment genes differentially expressed upon PA14 exposure between WT and skn-1gf animals, in comparison to OP50. B Representation of serotonin signaling pathways, including biosynthesis, uptake, and reuptake. C Selective representation of differentially expressed genes involved in serotonin signaling between WT and skn-1gf upon PA14 and OP50 exposure. D ELISA measurement of serotonin levels in whole animal extracts from WT and skn-1gf. E skn-1gf animals display enhanced sensitivity to treatment with the selective serotonin reuptake inhibitor fluoxetine. F Serotonin supplementation alleviates the apathy response of skn-1gf animals but only with continuous treatment from the earliest larval stage (G). Each of the food choice assay comprised of N ≥ 3 (with ≥ 150 worms per biological replicate per strain/condition) and analyzed via two-way ANOVA test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). For serotonin ELISA (N = 2; n = 2) were analyzed via a two-tailed/sided paired t test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). Individual “p” value numbers for the comparisons within each graph are placed above the respective comparison bars in the graph. All experiments were conducted at 25 °C. RNAseq data is available at the NIH (GEO) Gene Expression Omnibus (GSE251677). Source data for all Genotype-specific transcriptional responses are provided as a Source Data file 3. Source data for all behavioral responses are provided as a Source Data file 1.

Fig. 6. Pathogen apathy stems from systemic serotonin depletion.

Pathogen leaving behavior of (A) tph-1lf and (B) pah-1lf animals. C tph-1lf pah-1lf double mutants display pathogen apathy like skn-1gf mutants which is alleviated with supplementation of serotonin (D). Each of the food choice assays comprised of N ≥ 3 (with ≥ 150 worms per biological replicate per strain/condition) and analyzed via two-way ANOVA test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). Individual “p” value numbers for the comparisons within each graph is placed above the respective comparison bars in the graph. Source data for all behavioral responses are provided as a Source Data file 1.

Fig. 7. Serotonin depletion limits pathogen resistance.

Survival analysis of (A) WT and (B) skn-1gf animals with and without serotonin supplementation on PA14 fast kill assay plates. C Survival analysis of tph-1lf, pah-1lf, and tph-1lf pah-1lf on PA14-seeded fast kill assay plates. Each of the fast kill assays comprised N ≥ 3 (with ≥ 150 worms per biological replicate per strain/condition) and analyzed via two-way ANOVA test; **(p < 0.01) ***(p < 0.001) ****(p < 0.0001). Individual “p” value numbers for the comparisons within each graph are placed above the respective comparison bars in the graph. Source data for pathogen survival data are provided as a Source Data file 4.

Fig. 8. Model of pathogen apathy responses influenced by SKN-1 activity and serotonin.

SKN-1 activation results in limited serotonin bioavailability that drives pathogen apathy; a complex phenotype influenced by animal behavioral responses to microorganisms and regulated by both neurons and non-neuronal tissues.