Pharyngeal pumping inhibition and avoidance by acute exposure to high CO₂ levels are both regulated by the BAG neurons via different molecular pathways

Abstract

Carbon dioxide (CO₂) is a key molecule in many biological processes. Studies in humans, mice, D. melanogaster, C. elegans, unicellular organisms and plants have shed light on the molecular pathways activated by elevated levels of CO₂. However, the mechanisms that organisms use to sense and respond to high CO₂ levels remain largely unknown. Previous work has shown that C. elegans quickly avoid elevated CO₂ levels using mechanisms that involve the BAG, ASE and AFD neurons via cGMP- and calcium- signaling pathways. Here, we discuss our recent finding that exposure of C. elegans to high CO₂ levels leads to a very rapid cessation in the contraction of the pharynx muscles. Surprisingly, none of the tested CO₂ avoidance mutants affected the rapid pumping inhibition response to elevated CO₂ levels. A forward genetic screen identified that the hid-1-mediated pathway of dense core vesicle maturation regulates the pumping inhibition, probably through affecting neuropeptide secretion. Genetic studies and laser ablation experiments showed that the CO₂ response of the pharyngeal muscle pumping is regulated by the BAG neurons, the same neurons that mediate CO₂ avoidance.

Introduction

The respiratory gases, carbon dioxide (CO₂) and oxygen (O₂), are key molecules in oxidative metabolism. In order to maintain cellular homeostasis, all organisms must adapt to changes in the levels of these gases. The CO₂ and O₂ homeostasis in our body is mainly achieved by CO₂ chemoreceptors in the brain that promote respiratory responses to maintain normal CO₂ and O₂ levels in the blood. At the cellular level, under normal oxygen conditions, the transcription factor HIF-1α is hydroxylated in a conserved proline residue and subsequently targeted for proteasomal degradation. Under hypoxic conditions (low oxygen levels), HIF-1α is stabilized and orchestrates an adaptive response that maintains normal metabolism.¹

Whether a master regulator that responds to changes in CO₂ levels at the cellular level exists is not known. Several recent studies have shed new light on molecular pathways activated by elevated levels of CO₂. In lungs, elevated levels of CO₂ are associated with impaired fluid reabsorption as a consequence of Na,K- ATPase endocytosis.² The CO₂-mediated Na,K-ATPase endocytosis is partially regulated by activation of AMPK and subsequent activation of PKC-ζ.³ The soluble adenylyl cyclase (sAC) was also found to be activated by CO₂/HCO3.⁴ In a feedback loop found in alveolar epithelial cells, sAC activation results in elevated cAMP levels which leads to PKA-1α-dependent phosphorylation of the actin cytoskeleton component α-adducin and endocytosis of the Na,K-ATPase.⁵ CO₂ also inhibits cell proliferation by inducing mitochondrial dysfunction mediated by down regulation of the TCA cycle enzyme isocitrate dehydrogenase-2 (IDH2).⁶ In addition, elevated levels of CO₂ impair innate immunity responses in mammalian cells. CO₂ leads to nuclear translocation of RelB and IKKα, 2 central regulatory components of the NF-κB signaling pathway, which leads to significant attenuation of NF-kB signaling and altered inflammatory responses.^7,8 Most importantly, all the effects mentioned above are pH-independent, suggesting specific cellular responses to CO₂.

The nematode C. elegans, is a good model in which to study the physiological and molecular responses to high levels of CO₂.⁹ In this model organism, high levels of CO₂ induce an avoidance response, which is mediated by cGMP signaling pathway activation in the BAG neurons (Fig. 1).^10,11 This response is modulated by the nutritional state of the worm. Starved worms do not avoid CO₂ and worms mutated in insulin/IGF signaling, which mimics the starvation response, also do not avoid CO₂.^10,11 The homeostatic response to CO₂ is also modulated by both temperature and O₂ sensing neurons in that CO₂ is less aversive to animals acclimated to 15°C compared to animals acclimated to 22°C.¹² This difference requires the activation of temperature sensitive AFD neurons, which are also activated by CO₂. In addition, signaling from the oxygen-sensing neuron URX inhibits CO₂-mediated avoidance.^12,13 Little is known about how CO₂ is actually sensed in the BAG neurons, it was previously suggested that the guanylate cyclase receptor gcy-9, whose expression is directly controlled by the transcription factor ETS-5, serves as the CO₂ sensor in these neurons.¹⁴ ETS-5 is also required for proper differentiation and proper CO₂ responses of the BAG neurons.^15,16 More recently, EGL-13 and EGL-46 were also found to be important for the differentiation of the CO₂-sensing BAG neurons.^17,18

Figure 1.

Acute exposure of Caenorhabditis elegans to elevated CO₂ level causes animal avoidance and stops pharyngeal muscle contractions. Both responses are regulated by the BAG neurons but through separate signaling pathways. While avoidance requires cGMP signaling, the inhibition of muscle contraction in the pharynx is mediated by neuropeptide secretion. Both CO₂ responses are decreased following starvation.

Further studies are required to fully understand the molecular mechanisms induced by high levels of CO₂ and how these mechanisms regulate physiologic responses to elevated CO₂. We discuss here our recent study of a previously uncharacterized behavioral response of C. elegans to elevated CO₂, and a new component that is involved in C. elegans CO₂ signaling.¹⁹

High Levels of CO₂ Halt Pharynx Contractions

We previously described the effects of wild type C. elegans' exposure to chronic, high CO₂ levels.²⁰ These studies were performed to establish the potential use of C. elegans as a model organism for investigating the molecular mechanisms, at the whole organism level, that are activated in response to elevated levels of CO₂. We found that when wild type C. elegans are maintained at high CO₂ conditions they have a smaller brood size, delayed development, reduced motility that is coupled with striated muscle deterioration and a significant increase in life span. To gain a better insight into how worms respond to high levels of CO₂ we set to study the immediate responses elicited when worms are exposed to high CO₂ levels. We designed a small chamber, connected to a CO₂ tank, and used it to expose wild type C. elegans to elevated levels of CO₂ ranging from 5–20% CO₂.²¹ We noticed that in CO₂ concentrations higher than 10%, the pharynx, which normally contracts ∼200 times/min, almost completely stopped contracting after a few seconds of CO₂ exposure. The contraction cessation is probably counteracted by other response mechanisms since after 2 min of continuous exposure we started to observe a recovery in the pharynx response. However, even after prolonged exposure to high levels of CO₂ (30 min) a complete recovery of the pharynx contractions was not seen, suggesting a sustained response. Since CO₂ might potentially change the pH of the growth medium we also tested the response of the pharynx under different media conditions. The pharynx response was not affected by the growth medium pH, suggesting the effect of elevated CO₂ is probably not mediated by pH changes in the medium. It is well established that the nutritional state of the worm can significantly alter its responses to external environmental cues. Similarly, the nutritional state of the worm partially modulated the response of the pharynx to high levels of CO₂. In 10% CO₂, starved worms were able to partially contract the pharynx in contrast to well-fed animals where the pharynx contraction completely halted. However, in 20% CO₂ both starved and well-fed animals had a complete cessation of the pharynx contractions. This dose dependency suggests the existence of several response mechanisms that are activated at different CO₂ concentrations.

CO₂-Mediated Pumping Inhibition Regulation is not Shared with CO₂-Mediated Avoidance Components

We hypothesized that since the CO₂-mediated pumping inhibition is quick and robust, similar to the CO₂-mediated avoidance, the 2 behavioral responses probably share common components. However, all the CO₂ avoidance mutants that we tested showed a similar inhibition of the pumping in response to an acute high CO₂ exposure.¹⁹ The mutants that we tested included mutants in chemo sensation (tax-4), nutritional state of the worm (daf-2), ciliated neuron development (osm-3, che-10) and CO₂ neuron specification (gcy-9,ets-5). To identify new components that mediate the response of the pharynx to high CO₂, we performed a forward genetics screen after EMS mutagenesis. Specifically, we searched for mutants in which pumping inhibition is impaired when exposed to 10% CO₂. One of the genes identified in this screen was hid-1. HID-1 was previously identified in a screen for mutants that induce dauer formation under high temperature conditions.²² HID-1 was also found to be an important component of dense core vesicle secretion that controls neuropeptide release.^23-25 Interestingly, the response of this mutant to high CO₂ is dose dependent. After exposure to 5% CO₂, the pumping inhibition is completely rescued and pumping continues at the same rate as under normal atmospheric conditions. After exposure to 20% CO₂, this mutation can no longer rescue the CO₂-mediated pumping inhibition and pumping completely halts as in wild type worms.

Importantly, hid-1 is probably a component specifically involved in mediating the response of the pharynx to CO₂ but not of other responses induced by high CO₂. The development, fertility and brood size of hid-1 mutants are still impaired when chronically exposed to high levels of CO₂.

HID-1 functions in C. elegans to regulate neuropeptide secretion by dense core vesicles. We therefore investigated whether neuropeptide secretion constitutes a fundamental component of C. elegans’ response to acute high CO₂ exposure. We tested 2 mutants, unc-31, an essential player in the dense core vesicle secretion machinery and egl-21, which encodes a carboxypeptidase required for neuropeptide precursor processing. Like in hid-1, the response of these mutants to high CO₂ was significantly impaired compared to that of wild type worms. In contrast, unc-13 and rab-3 mutants (Fig. 1), specifically involved in synaptic vesicle secretion, responded to high CO₂ like the response of the wild type. Unfortunately, all of our attempts to identify single neuropeptide-encoding mutants in which the response to high CO₂ is impaired failed, suggesting that there is probably more than one neuropeptide/receptor involved in mediating this response.

HID-1 Function is Required in the BAG Neurons

hid-1 was previously shown to be expressed in the gut and also in the nerve system of C. elegans.^23,25 By tissue specifically expressing HID-1 in a hid-1 null background, either in the gut (under a ges-1 promoter) or in the nervous system (under a rab-3 promoter), we demonstrated that hid-1 is only needed in the nervous system to mediate pumping inhibition.¹⁹ We next searched for the neuronal subtype in which hid-1 mediates the effect of CO₂ on the pharynx. We used the nlp-3 promoter, which is expressed in neurons that secrete DCVs, to drive the expression of hid-1. Indeed, overexpression of hid-1 in this subset of neurons in a hid-1 null background restored the CO₂-mediated pumping inhibition to almost wild type levels. In addition, overexpression of hid-1 in the BAG neurons either under an flp-17 promoter or under a gcy-33 promoter restored the CO₂-medited pumping inhibition to wild type levels. Laser ablation of BAG neurons in hid-1-null strains overexpressing HID-1 under a gcy-33 or flp-17 promoter eliminated the CO₂-mediated pumping inhibition. This further supports that the expression of hid-1 is indeed required in BAG neurons. Our data support a model in which CO₂ elicits 2 different responses in the BAG neurons, the first induces avoidance and the second, mediated by hid-1, induces pumping inhibition.

Concluding Remarks

Studying C. elegans’ response to both chronic and acute high CO₂ levels has already yielded many insights into how cells react to this stressor and implications for patients suffering from pulmonary diseases. It is becoming clear that the nematode, Caenorhabditis elegans, presents a valuable model system to study the molecular sensing and response to changing levels of CO₂. This response involves specific neurons that sense the changes in CO₂ levels, neuropeptide secretion and activation of specific signaling pathways. Both the response and the recovery depend on the cell type and its metabolic state.

Many properties of organisms’ and cells’ response to elevated CO₂ levels have yet to be determined. In C. elegans, these include the identification of the sensing molecules, understanding why the BAG neurons require 2 different responding pathways that react to high CO₂ levels, discovering the responsible neuropeptides and their targets that respond to elevated CO₂ levels and how the adaption to longer and chronic exposures to elevated CO₂ levels occurs.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by NIH grant HL085534.