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Molecular mechanisms of acute oxygen sensing.

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

Periodo di rendicontazione: 2018-11-01 al 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 hypoxia. The grant application focused on four main projects:
Project 1. 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.
Project 2. Elucidating the properties of mitochondrial metabolism and biogenesis in O2-sensitive cells.
Project 3. Investigating the role of the adult neural stem cell niche in CB over-activation during sustained and intermittent hypoxia.
Project 4. Determining the impact of chemoreceptor inhibition -hypoxia intolerance- on adult brain neurogenesis.
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. Using transformed mouse CB chemoreceptor cells (glomus cells) we have generated several immortalized cell lines with variable sensitivity to hypoxia (high, medium, low). We have already characterized the O2-sensing phenotype of these cells and, currently, we are beginning to analyze whether the different physiological responses are paralleled by specific changes in the expression of the genes relevant for oxygen sensing.
c.2. Generation of a mouse model with inducible overexpression of Phd3. As we have found Phd3 mRNA levels to be particularly low in CB glomus cells, we have started the generation of a conditional mouse model with overexpression of the O2-sensitive enzyme. We have already finished the molecular biology and prepared the donor plasmid for injection into zygotes using CRISPR-Cas9 technologies. We expect to have the first animals to do experiments by the end of the year.
c.3. Role of Hif2a down regulation in acute oxygen sensing. As Hif2a is constitutively expressed in CB glomus cells, we have studied whether this transcription factor has any role in acute O2-sensing. The experiments were done using conditional Epas1 (Hif2a) knockout mice. We have found that inducible Hif2a-deficiency in adult mice results in abolition of the HVR as well as the cellular responses to hypoxia in CB glomus cells, although responsiveness to hypercapnia remains unaltered. The loss of Hif2a leads to a selective down-regulation of the atypical mitochondrial subunits (Ndufa4l2, Cox4i2 and Cox8b) in glomus cells. Currently we are in the final experimental stages of this project, and we expect to submit a manuscript for publication before the end of the year.
c.4. Role of atypical mitochondrial subunits in acute oxygen sensing. The role of the three atypical mitochondrial subunits found over-expressed in CB glomus cells is being studied in genetically modified mice. Conditional knockouts for Ndufa4l2 and Cox4i2 were already available and are currently studied in our laboratory. We have started the generation of the conditional Cox8b knockout and we expect it to be available for experiments at the beginning of year 2019.
c.5. Generation of a mouse model with inducible deletion of the Pcx gene. The role of Pcx, found over-expressed in CB glomus cells, is being studied in genetically modified mice. We have started the generation of the conditional Pcx knockout mice and we expect them to be available for experiments within the next few months.

d) Project 3 in the application (Investigating the role of the adult neural stem cell niche in CB over-activation during sustained and intermittent hypoxia).
We have made important advances in this project thanks to the identification in the adult rat CB of a subpopulation of TH-positive cells, which are “immature” (electrically excitable but O2-insensitive) that upon exposure to sustained hypoxia are rapidly converted to mature O2-sensitive glomus cells (see Sobrino et al., EMBO Reports, 2018). In most aspects, this subpopulation of premature glomus cells behaves in a way similar to neuroblasts (precursors of mature neurons) in the central germinal centers. Interestingly, we have preliminary data indicating that similar changes occur in the CB of rats subjected to chronic intermittent hypoxia. If these findings are confirmed, they could represent a mechanistic explanation for CB over-activation (increased sympathetic outflow) in sleep apnea patients.

e) Project 4 in the application (Determining the impact of chemoreceptor inhibition -hypoxia intolerance- on adult brain neurogenesis).
In collaboration with Dr. David Macias (group of Dr. Randy Johnson at the University of Cambridge, UK) we have developed a model of CB atrophy (lack of CB development) based on the embryonic ablation of the Hif2a gene. These animals show intolerance to hypoxia (Macias et al., ELife, 2018). Based on this model we plan to investigate in the future the changes in central neurogenesis in normoxic and hypoxic conditions.
In the context of this project, we have also studied the effect of genetic inhibition of MCI activity on the proliferation and differentiation of central neural progenitors. We have found that MCI is not needed for the maintenance of neural stem cells but it is required for their proliferation and differentiation to oligodendrocytes and neurons. We are currently preparing a manuscript with these data, which we expect to submit for publication within the next few months.
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.