In multicellular organisms, signaling from the nervous system to the peripheral tissues can activate physiological responses to stress. Here, we show that inter-tissue stress communication can also function in reverse, i.e. from the peripheral tissue to the nervous system. osm-8 mutants, which activate the physiological osmotic stress response in the C. elegans skin, also exhibit defective osmotic avoidance (Osm) behavior, via a direct and specific effect on ASH osmosensory neuron excitability. Both osm-8 and the Patched-related gene ptr-23, mutations in which suppress all osm-8 phenotypes, function in the hypodermal lysosomes to regulate both physiology and behavior. Unbiased lipidomics shows that osm-8 leads to a ptr-23-dependent elevation of the lysosome specific lipid bis(monoacylglycero)phosphate (BMP) and expansion of the pool of hypodermal lysosomes. Just as genetic activation of the osmotic stress response by loss of osm-8 in the hypodermis causes an Osm phenotype, acute physiological exposure to osmotic stress also confers a reversible Osm phenotype. Behavioral and genetic plasticity requires biosynthesis of the compatible solute glycerol, a key physiological output of the organismal osmotic stress response. However, ptr-23 is only required for osm-8 induced behavioral plasticity and not physiological plasticity. Instead, both genetic and physiologically induced Osm phenotypes require the unusual non-neuronal lysosomal V-ATPase subunit vha-5, which is also critical for organismal osmotic stress survival. Together, these data reveal that genetic or physiological activation of stress signaling from the skin elicits lysosome-associated signals that modulate organismal neurophysiology to attenuate a sensory neuron circuit. Such ‘body-brain’ interoceptive communication may allow organisms to better match neuronal decision-making with organismal physiological state.
Cellular responses to alterations in homeostasis, such as changes in temperature, oxygen, and osmolarity, are commonly referred to as cellular stress responses. This is because cells contain autonomous mechanisms for sensing, signaling, and adapting to the particular challenges posed by each individual stressor. However, in an organismal setting, these autonomous cellular stress response pathways become subservient to systemic control mechanisms. For example, in mice, genetic or pharmacological activation of autophagy in the hypothalamic POMC neurons leads to activation of autophagy in peripheral tissues such as the liver and adipose cells. In C. elegans, genetic activation of the mitochondrial or endoplasmic reticulum stress response in neurons leads to activation of these same stress response pathways in peripheral intestinal cells. Similarly, optogenetic activation of temperature-sensing neurons can activate the heat shock transcription factor in the germline. While this neuron-to-soma control of stress response pathways is now well established, what is not clear is whether or not information flows in the reverse direction, i.e. from soma to neurons. Some studies suggest that food and/or pathogen based stress signaling from epithelial cells in C. elegans can signal to neurons to modify physiological states, such as longevity, sleep, and learned pathogen avoidance. Whether or not abiotic factors, such as osmolarity, also modify behavior through changes in physiological state is less well understood.
The nematode C. elegans exhibits two robust responses to increased environmental osmolarity, i.e. hypertonic stress. First, C. elegans exhibit behavioral avoidance to hypertonicity. This response is driven by a well-described neuronal avoidance circuit made up of the amphid and phasmid sensory neurons ASH and PHB which signal to the command interneuron AVA to initiate backwards motion and reorientation away from the stressor. Second, when worms are unable to avoid hypertonicity, they activate physiological adaptation pathways, primarily in the hypodermis. This includes transcriptional up- and down-regulation of >300 osmotic response genes, such as the glycerol biosynthetic enzyme gpdh-1 via the activity of hypodermal molecular signaling pathways. Glycerol accumulation closely tracks extracellular solute levels which allows C. elegans to maintain cell and tissue volume without raising cytoplasmic ionic strength. Currently, the connection between osmotic avoidance behavior and peripheral tissue osmotic stress responses are not clear. ASH osmo-sensing does not appear to be required for hypertonicity induced gpdh-1 upregulation or glycerol accumulation in the somatic tissues. Whether or not somatic stress signaling modifies ASH osmotic avoidance behavior has not been investigated.
Upregulation of the glycerol biosynthetic enzyme gpdh- 1 in the epithelial tissues is a robust and specific marker of the physiological response to hypertonic stress in C. elegans. gpdh-1 upregulation by hypertonicity is positively regulated by the O-GlcNAc transferase ogt-1 and the polyadenylation and cleavage factor complex subunits such as Symplekin/symk-1. Unexpectedly, gpdh-1 is also under extensive negative regulation, as inhibition of >100 genes cause significant upregulation of gpdh-1 under non-stressful isotonic conditions. These negative regulators result in a physiological state change, as mutant animals become completely resistant to dehydration when exposed to extreme hypertonicity (>500mM NaCl) and accumulate the osmolyte glycerol at close to molar levels. One of these negative regulators is osm-8, a 331 residue protein with a signal sequence and S/T-rich repeats. Loss of osm-8 leads to upregulation of gpdh-1, as well as most of the ORG transcription program, and accumulation of glycerol. As a result, >90% of osm-8 mutants retain mobility in >500mM NaCl, a condition in which 0% of wild type animals are motile. Through a genetic suppressor screen, we found that all of these physiological phenotypes require the Nieman-Pick C1 (NPC1)/patched-related (ptr) receptor ptr-23. Tissue-specific and temporal rescue experiments reveal that osm-8 is required in the hypodermal epithelial cells during the L4 stage for these phenotypes. If and how osm-8 and ptr-23 regulate neuronal osmotic avoidance behavior has not been determined.
Here, we show that, in addition to its role in regulating peripheral physiology, osm-8 also regulates neuronal osmotic avoidance behavior by reducing the sensitivity of the ASH neuron to hypertonic stimuli. osm-8 behavioral defects are completely dependent on ptr-23. Furthermore, the expression of osm-8 and ptr-23 in the hypodermis is necessary and sufficient to regulate neuronal behavior. Endogenously tagged alleles show that OSM-8 and PTR-23 co-localize to the hypodermal lysosomes. Unbiased lipidomic analysis shows that the lysosome-specific lipid bis(monoacylglycero)phosphate (BMP) exhibits ptr-23-dependent upregulation in osm-8 mutants, which results in an expansion of the pool of lysosomes in the hypodermis. Acute adaptation of wild type worms to mild hypertonicity also induces a reversible osmotic avoidance defective phenotype like osm-8. The acquired Osm phenotype is dependent on both glycerol biosynthesis enzymes gpdh-1 and gpdh-2 and the lysosomal V-ATPase subunit vha-5, which is also required for osm-8 behavioral phenotypes. Together, these data suggest that the osm-8-ptr-23-vha-5 pathway that regulates peripheral stress physiology in the skin also modulates ASH-driven behavioral sensitivity to osmotic stress, thus synchronizing behavioral and physiological states at the organismal level.
The single osm-8 allele, n1518, was previously shown to constitutively upregulate the glycerol biosynthesis gene gpdh-1, contain elevated glycerol levels, and exhibit an osmotic stress resistance (Osr) phenotype to 500 mM NaCl, a normally lethal hypertonic stress to naïve wild type animals. All of these phenotypes are dependent on the function of the patched-related receptor ptr-23. To create definitive null alleles for these genes, we generated deletion alleles for both osm-8 and ptr-23 using CRISPR/Cas9. Similar to the originally reported alleles, the osm-8(dr170) allele was also Osr while the ptr-23(dr180) allele exhibited no Osr phenotype on its own but fully suppressed the Osr phenotype of osm-8(d170). Mutations in neuron-expressed Osm genes, such as osm-6, do not exhibit an Osr phenotype. These new alleles represent definitive null alleles for both osm-8 and ptr-23 that phenocopy previously described mutants and were used for all subsequent studies, unless otherwise indicated.
Figure 1 osm-8 physiological and behavioral phenotypes depend on ptr-23.
A) Diagram of the osm-8 and ptr-23 alleles generated in this study. A detailed list of these and other strains can be found in Table 1. The osm-8(n1518) allele was characterized in a prior study. B) Osmotic stress resistance (OSR) phenotype of the indicated genotypes. N=8 replicates per genotype (10 animals per replicate, N=80 per genotype). ****-p<0.0001, One-way ANOVA with Kruskal-Wallis post hoc test. Individual data points are shown along with the mean ± S.D. C) Osmotic avoidance phenotype of the indicated genotypes. N=8 replicates per genotype (10 animals per replicate, N=80 per genotype). ***-p<0.001, ‘ns’ – p>0.05, One-way ANOVA with Kruskal-Wallis post hoc test. Individual data points are shown along with the mean ± S.D. D) Osmotic avoidance phenotype of the indicated genotypes. N=8 replicates per genotype (10 animals per replicate, N=80 per genotype). **-p<0.01, ‘ns’ – p>0.05, One-way ANOVA with Kruskal-Wallis post hoc test. Individual data points are shown along with the mean ± S.D.
While osm-8 is annotated to exhibit Osmotic Avoidance (Osm) behavioral defects, these phenotypes have never been described or published. When C. elegans encounter hypertonic environments, they first attempt to avoid the stimuli through a well characterized behavioral avoidance response.
