Regulator of Lipid Metabolism NHR-49 Mediates Pathogen Avoidance through Precise Control of Neuronal Activity

Abstract

Precise control of neuronal activity is crucial for the proper functioning of neurons. How lipid homeostasis contributes to neuronal activity and how much of it is regulated by cells autonomously is unclear. In this study, we discovered that absence of the lipid regulator nhr-49, a functional ortholog of the peroxisome proliferator-activated receptor (PPAR) in Caenorhabditis elegans, resulted in defective pathogen avoidance behavior against Pseudomonas aeruginosa (PA14). Functional NHR-49 was required in the neurons, and more specifically, in a set of oxygen-sensing body cavity neurons, URX, AQR, and PQR. We found that lowering the neuronal activity of the body cavity neurons improved avoidance in nhr-49 mutants. Calcium imaging in URX neurons showed that nhr-49 mutants displayed longer-lasting calcium transients in response to an O₂ upshift, suggesting that excess neuronal activity leads to avoidance defects. Cell-specific rescue of NHR-49 in the body cavity neurons was sufficient to improve pathogen avoidance, as well as URX neuron calcium kinetics. Supplementation with oleic acid also improved avoidance behavior and URX calcium kinetics, suggesting that the defective calcium response in the neuron is due to lipid dysfunction. These findings highlight the role of cell-autonomous lipid regulation in neuronal physiology and immune behavior.

Keywords: Caenorhabditis elegans; PPAR; Pseudomonas aeruginosa; immune behavior; lipid metabolism; neuron.

Figure 1

nhr-49 mutants exhibit decreased survival and defective pathogenic lawn avoidance behavior against PA14. (A–C) Representative survival plots in small lawns. Three trials were conducted for all survival assays (See Figure S3 and Table S3 for additional trials). (D) After 5 h of PA14 exposure, wild-type worms are seen outside the lawn (arrowhead), whereas nhr-49 mutants remain inside the lawn. (E) Lawn avoidance of N2 and nhr-49. The dotted gray line at the 8 h mark indicates that statistical analyses were conducted using the data for 8 h post-exposure. (F) Representative survival plots of big lawn survival assay (See Figure S3 and Table S3 for additional trials). (G–I) Lawn avoidance of tissue-specific transgenic strains. The dotted gray line at the 8 h mark indicates that statistical analyses were conducted using the data for 8 h post-exposure. For statistical analyses of avoidance assays, each strain was compared to nhr-49 (one-way ANOVA with Dunnett’s multiple comparisons test). Asterisks indicate the p-value (* p < 0.05; ** p < 0.01).

Figure 2

NHR-49 in cholinergic and glutamatergic neurons promote pathogenic lawn avoidance. Lawn avoidance of serotonergic (A), dopaminergic (B), cholinergic (C), and glutamatergic (D) neuron rescue strains. (E,F) Representative survival plots for cholinergic and glutamatergic rescue strains. Plate with small green lawn indicates survival was tested in a small lawn assay. Three trials were conducted for all survival assays (See Figure S3 and Table S3 for additional trials). For lawn avoidance (A–D), the dotted gray line at the 8 h mark indicates that statistical analyses were conducted using the data for 8 h post-exposure. Each strain was compared to nhr-49. (one-way ANOVA with Dunnett’s multiple comparisons test). Asterisks indicate the p-value (* p < 0.05; ** p < 0.01).

Figure 3

NHR-49 in the URX, AQR, and PQR body cavity neurons is sufficient to promote avoidance. (A) Signaling pathways involved in promoting pathogenic lawn avoidance. (B) Real-time PCR quantification of daf-7 mRNA expression after 10 h of PA14 exposure. (C) The lawn avoidance defect of nhr-49 is not suppressed by daf-3 mutations. (D) Real-time PCR quantification of NPR-1 ligands flp-18 and flp-21 mRNA expression after 10 h of PA14 exposure. (E,F) Body cavity neuron-specific rescue improves lawn avoidance (E) and survival (F). Three trials were conducted for survival (see Figure S3 and Table S3 for additional trials). (G–I) Lawn avoidance in strains with body cavity neuron-specific expression of nhr-49 gain-of-function alleles. The dotted gray line at the 8 h mark indicates that statistical analyses were conducted using the data for 8 h post-exposure. Each strain was compared to nhr-49, except (C), which was compared with daf-3 (one-way ANOVA with Dunnett’s multiple comparisons test). Asterisks indicate the p-value (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 4

Decreased activity or ablation of body cavity neurons leads to improved avoidance in nhr-49 mutants. (A) PA14 avoidance in 21% and 10% ambient oxygen after 8 h. (B) Pathogen avoidance of oxygen-sensing defective mutants. (C) Genetic ablation of body cavity neurons results in improved avoidance in both wild-type and nhr-49 mutants. (D) Abolishing the synaptic signal does not improve avoidance. The dotted gray line at the 8 h mark indicates that statistical analyses were conducted using the data for 8 h post-exposure. Each strain was compared to nhr-49 (one-way ANOVA with Dunnett’s multiple comparisons test for (B); Student’s t-test for (C,D)). Asterisks indicate the p-value (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

Calcium transients of nhr-49 show prolonged signals. (A–C) Calcium imaging results of the N2, nhr-49, and body cavity rescue strains in response to an O₂ upshift from 10% to 21%. (D–I) Detailed characterization of calcium transients. Peak amplitude (E), half time of rise and decay (F,G), area under the curve (H), and transient duration at 10, 50, and 80% repolarization (I) were assessed. (J) Avoidance behavior of N2 and nhr-49 mutants supplemented with 300 μM OA. The dotted gray line at the 8 h mark indicates that statistical analyses were conducted using the data at this time point. (K) Calcium imaging results of nhr-49 supplemented with OA. Asterisks indicate the p-value (* p < 0.05; ** p < 0.01), as determined by the one-way ANOVA with Dunnett’s multiple comparisons test for (E–I) and the Student’s t-test for (J).