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Molecular mechanisms of acute oxygen sensing.

Periodic Reporting for period 3 - OxygenSensing (Molecular mechanisms of acute oxygen sensing.)

Reporting period: 2018-11-01 to 2020-04-30

Achievements of the project
Oxygen (O2) is essential for mammalian homeostasis and, therefore, the provision of sufficient O2 to the tissues is a major physiological challenge. However, the mechanisms whereby cells detect changes in O2 tension (PO2) are not well understood. The lack of O2 (hypoxia) induces reflex adaptive responses (hyperventilation and sympathetic activation), which in seconds increase the uptake of O2 from the atmosphere and its distribution to the tissues of the organism. These responses are mediated by the peripheral chemoreceptors, in particular the carotid body (CB), which contains neuron-like O2-sensitive cells (glomus cells) that in response to hypoxia release transmitters to activate sensory nerve fibers impinging upon the brain respiratory and autonomic centers. The basic mechanisms underlying how the specialized chemoreceptor cells are able to “sense” rapid changes in O2 in the environment have remained elusive. Since the beginning of the project we have significantly advanced in the identification of the mechanisms of responsiveness to hypoxia by peripheral chemoreceptor cells, which was the main objective of the ERC grant. We have reported a gene expression profile characteristic of acute O2 sensing cells. This includes several atypical mitochondrial electron transport complex IV (MCIV) subunits, metabolic enzymes, hypoxia inducible factor 2 and several ion channel subtypes. We have proposed the most complete model to date of acute O2 sensing. It suggests that slow down of mitochondrial electron transport during hypoxia leads to the accumulation of reduced ubiquinone and the production of NADH and reactive oxygen species (ROS) in mitochondrial complex I (MCI). These signaling molecules modulate ion channel function to induce cell depolarization, Ca2+ influx and transmitter release. In addition, we have advanced in the understanding of the properties of the CB neurogenic niche that helps to explain CB plasticity during exposure to either sustained or intermittent chronic hypoxia. We have discovered a new population of CB “neuroblasts” that in few hours can divide and differentiate into mature O2-sensitive glomus cells. Finally, we have been able to generate a genetic mouse model of CB developmental atrophy, which exhibits lack of responsiveness to hypoxia. We plan to use this mouse model in future experiments to investigate the impact of hypoxia intolerance on central neurogenesis.

Summary of the context and overall objectives of the project
O2 is essential for most eukaryotic cells as it is used as a general acceptor of electrons in oxidative phosphorylation. However, the lack of O2 (hypoxia) is a relatively frequent condition in animals and humans living at high altitude or suffering diseases that compromise gas exchange in the lungs. Sustained hypoxia is known to induce in cells an adaptive gene expression program, due to the activation of the prolyl hydroxylase/hypoxia inducible factors pathway, which activates erythropoiesis, angiogenesis and glycolysis. However, immediate survival of animals upon exposure to low O2 tension requires fast activation (in seconds) of cardiorespiratory reflexes to increase O2 uptake and its distribution to the most demanding tissues (such as brain or heart). These acute responses to hypoxia depend on the activation of peripheral chemoreceptors, in particular the CB, and other organs forming the homeostatic acute O2 sensing system. The CB contains O2 sensitive glomus cells, which depolarize in response to hypoxia and release transmitters to activate sensory fibers conveying the information to the respiratory and autonomic centers in the brainstem. In spite of the fundamental biomedical importance of acute O2 sensing, the underlying molecular mechanisms have remained elusive. The general objective of the ERC grant was to unveil the mechanisms of acute O2 sensing and to identify the nature of the plastic changes occurring in the CB during adaptation to hyp
The main results achieved so far are:

a) Project 1 in the application (Determining the role mitochondria, particularly the role of mitochondria complex I, in acute O2 sensing by arterial chemoreceptors and other cells that respond acutely to hypoxia).
We have done important advances in this project, which we consider one of the foundations of the general research proposal. Coincident with the onset of the project, in October 2015, we published a model of acute oxygen sensing proposing a fundamental role of MCI in acute responses to hypoxia by arterial chemoreceptors cells in the CB and the adrenal medulla (AM) (Fernández-Agüera et al., Cell Metabolism, 2015). In the last two years we have made important advances in this project and performed experiments on genetically modified mice with conditional ablation of the Ndufs2 gene, which encodes a protein forming the ubiquinone-binding site at MCI. Our data have served to postulate a comprehensive model of acute O2 sensing in which accumulation of reduced ubiquinone during hypoxia determines the slow down (or even reversion) of MCI electron flow and the production of ROS and accumulation of NADH. These molecules signal the membrane K+ channels to induce cell depolarization and release of transmitters, which activate the respiratory center to elicit the hypoxic ventilatory response (HVR). These data, which were by themselves a main goal of the project, have appeared on a manuscript accepted in Cell Metabolism during the last days of April 2018 (Arias-Mayenco et al., Cell Metabolism, 2018).

b) Project 2 in the application (Elucidating the properties of mitochondrial metabolism and biogenesis in O2-sensitive cells).
In the context of this project, we have performed a comparative gene expression analysis of CB, AM and superior cervical ganglion (SCG) cells. These three cell types have a common embryological origin but different sensitivity to hypoxia, being the CB and AM oxygen sensitive (CB>AM) and SCG oxygen insensitive. We have found several genes that define a “metabolic signature profile” in acute oxygen sensing cells. These genes encode three atypical mitochondrial subunits (Ndufa4l2, Cox8b and Cox4i2), HIF2a, pyruvate carboxylase (Pcx), and several ion channel subunits (Task1, Task 3, a1H T-type Ca2+ channel, and TrpC5) which are up-regulated, and Phd3 which is down-regulated (see Gao et al., Journal of Physiology, 2017). We have also found that biotin, a cofactor necessary for Pcx activity, is highly concentrated in CB cells (Ortega-Sáenz et al., Journal of Physiology, 2016). Although none of these genes may be absolutely required for acute O2-sensing, we think that, together, they generate the appropriate metabolic status to confer upon the chemoreceptor cells special sensitivity to changes in PO2. One of the most important consequences of the identification of the “genetic profile” characteristic of acutely O2-sensing cells, is that it can be tested experimentally using genetically modified animal models. Indeed, the role of each of these genes is being studied in ongoing experiments briefly described in the next section.
In addition to the genes mentioned in the preceding paragraph, one of the genes more highly expressed in the CB glomus cells is Olfr78, encoding an atypical G-protein coupled olfactory receptor expressed in several tissues outside the olfactory epithelium. Surprisingly, Olfr78 has been suggested to be a lactate receptor essential for the CB-dependent oxygen regulation of breathing. However, this proposal has been challenged by our experiments in collaboration with the groups of Peter Mombaerts (Max-Plank Institute, Frankfurt, Germany) Randy Johnson (University of Cambridge, UK) and Hiro Matsunami (Duke University, USA) showing that Olfr78 is not necessary for sensing hypoxia or lactate (see Torres-Torrelo et al., Nature, 2018).

c) Ongoing experiments related to projects 1 and 2.
c.1. CB cell lines with variable sensitivity to hypoxia
Acute oxygen sensing is essential for life in mammals. However understanding the underlying molecular mechanism has remained elusive. The progress reached in the current ERC grant, has permitted us to elaborate the most advanced and comprehensive model of acute oxygen sensing available so far. We think that the recently published Cell Metabolism paper (Arias-Mayenco et al, 2018) represents a breakthrough in the field regarding an issue that has been investigated by us, and others, for the last 30 years. An additional milestone is the comparative gene expression analysis between oxygen sensitive (cells in CB and AM) and oxygen insensitive (SCG neurons) sympatho-adrenal cells, which has allowed us to propose the existence of a “signature metabolic profile” in acute O2-sensing cells (Gao et al., Journal of Physiology, 2017).
As indicated in the preceding section, we are currently testing experimentally the main details of the proposed model of acute O2 sensing in CB glomus cells. During the remaining period of the grant, we also plan to explore if the model of CB O2 sensing is applicable to other O2 sensitive cells (in particular systemic and pulmonary arterial myocytes). We also plan to continue with the already started research on the plastic changes occurring in the CB and AM during chronic intermittent hypoxia.