Carotid Body Function in Tyrosine Hydroxylase Conditional Olfr78 Knockout Mice.

The Olfr78 gene encodes a G-protein-coupled olfactory receptor that is expressed in several ectopic sites. Olfr78 is one of the most abundant mRNA species in carotid body (CB) glomus cells. These cells are the prototypical oxygen (O2) sensitive arterial chemoreceptors, which, in response to lowered O2 tension (hypoxia), activate the respiratory centers to induce hyperventilation. It has been proposed that Olfr78 is a lactate receptor and that glomus cell activation by the increase in blood lactate mediates the hypoxic ventilatory response (HVR). However, this proposal has been challenged by several groups showing that Olfr78 is not a physiologically relevant lactate receptor and that the O2-based regulation of breathing is not affected in constitutive Olfr78 knockout mice. In another study, constitutive Olfr78 knockout mice were reported to have altered systemic and CB responses to mild hypoxia. To further characterize the functional role of Olfr78 in CB glomus cells, we here generated a conditional Olfr78 knockout mouse strain and then restricted the knockout to glomus cells and other catecholaminergic cells by crossing with a tyrosine hydroxylase-specific Cre driver strain (TH-Olfr78 KO mice). We find that TH-Olfr78 KO mice have a normal HVR. Interestingly, glomus cells of TH-Olfr78 KO mice exhibit molecular and electrophysiological alterations as well as a reduced dopamine content in secretory vesicles and neurosecretory activity. These functional characteristics resemble those of CB neuroblasts in wild-type mice. We suggest that, although Olfr78 is not essential for CB O2 sensing, activation of Olfr78-dependent pathways is required for maturation of glomus cells.

Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Constitutive Expression of Hif2α Confers Acute O2 Sensitivity to Carotid Body Glomus Cells.

Acute oxygen (O2) sensing and adaptation to hypoxia are essential for physiological homeostasis. The prototypical acute O2 sensing organ is the carotid body, which contains chemosensory glomus cells expressing O2-sensitive K+ channels. Inhibition of these channels during hypoxia leads to cell depolarization, transmitter release, and activation of afferent sensory fibers terminating in the brain stem respiratory and autonomic centers. Focusing on recent data, here we discuss the special sensitivity of glomus cell mitochondria to changes in O2 tension due to Hif2α-dependent expression of several atypical mitochondrial electron transport chain subunits and enzymes. These are responsible for an accelerated oxidative metabolism and the strict dependence of mitochondrial complex IV activity on O2 availability. We report that ablation of Epas1 (the gene coding Hif2α) causes a selective downregulation of the atypical mitochondrial genes and a strong inhibition of glomus cell acute responsiveness to hypoxia. Our observations indicate that Hif2α expression is required for the characteristic metabolic profile of glomus cells and provide a mechanistic explanation for the acute O2 regulation of breathing.

Transgenic NADH dehydrogenase restores oxygen regulation of breathing in mitochondrial complex I-deficient mice.

The hypoxic ventilatory response (HVR) is a life-saving reflex, triggered by the activation of chemoreceptor glomus cells in the carotid body (CB) connected with the brainstem respiratory center. The molecular mechanisms underlying glomus cell acute oxygen (O2) sensing are unclear. Genetic disruption of mitochondrial complex I (MCI) selectively abolishes the HVR and glomus cell responsiveness to hypoxia. However, it is unknown what functions of MCI (metabolic, proton transport, or signaling) are essential for O2 sensing. Here we show that transgenic mitochondrial expression of NDI1, a single-molecule yeast NADH/quinone oxidoreductase that does not directly contribute to proton pumping, fully recovers the HVR and glomus cell sensitivity to hypoxia in MCI-deficient mice. Therefore, maintenance of mitochondrial NADH dehydrogenase activity and the electron transport chain are absolutely necessary for O2-dependent regulation of breathing. NDI1 expression also rescues other systemic defects caused by MCI deficiency. These data explain the role of MCI in acute O2 sensing by arterial chemoreceptors and demonstrate the optimal recovery of complex organismal functions by gene therapy.

Protection and Repair of the Nigrostriatal Pathway with Stem-Cell-Derived Carotid Body Glomus Cell Transplants in Chronic MPTP Parkinsonian Model.

Antiparkinsonian carotid body (CB) cell therapy has been proven to be effective in rodent and nonhuman primate models of Parkinson's disease (PD), exerting trophic protection and restoration of the dopaminergic nigrostriatal pathway. These neurotrophic actions are mediated through the release of high levels of glial-cell-line-derived neurotrophic factor (GDNF) by the CB transplant. Pilot clinical trials have also shown that CB autotransplantation can improve motor symptoms in PD patients, although its effectiveness is affected by the scarcity of the grafted tissue. Here, we analyzed the antiparkinsonian efficacy of in vitro-expanded CB dopaminergic glomus cells. Intrastriatal xenografts of rat CB neurospheres were shown to protect nigral neurons from degeneration in a chronic MPTP mouse PD model. In addition, grafts performed at the end of the neurotoxic treatment resulted in the repair of striatal dopaminergic terminals through axonal sprouting. Interestingly, both neuroprotective and reparative effects induced by in vitro-expanded CB cells were similar to those previously reported by the use of CB transplants. This action could be explained because stem-cell-derived CB neurospheres produce similar amounts of GDNF compared to native CB tissue. This study provides the first evidence that in vitro-expanded CB cells could be a clinical option for cell therapy in PD.

Acute oxygen sensing by vascular smooth muscle cells.

An adequate supply of oxygen (O2) is essential for most life forms on earth, making the delivery of appropriate levels of O2 to tissues a fundamental physiological challenge. When O2 levels in the alveoli and/or blood are low, compensatory adaptive reflexes are produced that increase the uptake of O2 and its distribution to tissues within a few seconds. This paper analyzes the most important acute vasomotor responses to lack of O2 (hypoxia): hypoxic pulmonary vasoconstriction (HPV) and hypoxic vasodilation (HVD). HPV affects distal pulmonary (resistance) arteries, with its homeostatic role being to divert blood to well ventilated alveoli to thereby optimize the ventilation/perfusion ratio. HVD is produced in most systemic arteries, in particular in the skeletal muscle, coronary, and cerebral circulations, to increase blood supply to poorly oxygenated tissues. Although vasomotor responses to hypoxia are modulated by endothelial factors and autonomic innervation, it is well established that arterial smooth muscle cells contain an acute O2 sensing system capable of detecting changes in O2 tension and to signal membrane ion channels, which in turn regulate cytosolic Ca2+ levels and myocyte contraction. Here, we summarize current knowledge on the nature of O2 sensing and signaling systems underlying acute vasomotor responses to hypoxia. We also discuss similarities and differences existing in O2 sensors and effectors in the various arterial territories.

Rearrangement of cell types in the rat carotid body neurogenic niche induced by chronic intermittent hypoxia.

The carotid body (CB) is a prototypical acute oxygen (O2 )-sensing organ that mediates reflex hyperventilation and increased cardiac output in response to hypoxaemia. CB overactivation, secondary to the repeated stimulation produced by the recurrent episodes of intermittent hypoxia, is believed to contribute to the pathogenesis of sympathetic hyperactivity present in sleep apnoea patients. Although CB functional plasticity induced by chronic intermittent hypoxia (CIH) has been demonstrated, the underlying mechanisms are not fully elucidated. Here, we show that CIH induces a small increase in CB volume and rearrangement of cell types in the CB, characterized by a mobilization of immature quiescent neuroblasts, which enter a process of differentiation into mature, O2 -sensing and neuron-like, chemoreceptor glomus cells. Prospective isolation of individual cell classes has allowed us to show that maturation of CB neuroblasts is paralleled by an upregulation in the expression of specific glomus cell genes involved in acute O2 -sensing. CIH enhances mitochondrial responsiveness to hypoxia in maturing neuroblasts as well as in glomus cells. These data provide novel perspectives on the pathogenesis of CB-mediated sympathetic overflow that may lead to the development of new pharmacological strategies of potential applicability in sleep apnoea patients. KEY POINTS: Obstructive sleep apnoea is a frequent condition in the human population that predisposes to severe cardiovascular and metabolic alterations. Activation of the carotid body, the main arterial oxygen-sensing chemoreceptor, by repeated episodes of hypoxaemia induces exacerbation of the carotid body-mediated chemoreflex and contributes to sympathetic overflow characteristic of sleep apnoea patients. In rats, chronic intermittent hypoxaemia induces fast neurogenesis in the carotid body with rapid activation of neuroblasts, which enter a process of proliferation and maturation into O2 -sensing chemoreceptor glomus cells. Maturing carotid body neuroblasts and glomus cells exposed to chronic intermittent hypoxia upregulate genes involved in acute O2 sensing and enhance mitochondrial responsiveness to hypoxia. These findings provide novel perspectives on the pathogenesis of carotid body-mediated sympathetic hyperactivation. Pharmacological modulation of carotid body fast neurogenesis could help to ameliorate the deleterious effects of chronic intermittent hypoxaemia in sleep apnoea patients.

Full protection from SARS-CoV-2 brain infection and damage in susceptible transgenic mice conferred by MVA-CoV2-S vaccine candidate.

Vaccines against SARS-CoV-2 have been shown to be safe and effective but their protective efficacy against infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse model of severe coronavirus disease 2019 (COVID-19), we report a spatiotemporal description of SARS-CoV-2 infection and replication through the brain. SARS-CoV-2 brain replication occurs primarily in neurons, leading to neuronal loss, signs of glial activation and vascular damage in mice infected with SARS-CoV-2. One or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. These findings further support the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19.

Oxygen regulation of breathing is abolished in mitochondrial complex III-deficient arterial chemoreceptors.

Acute oxygen (O2) sensing is essential for adaptation of organisms to hypoxic environments or medical conditions with restricted exchange of gases in the lung. The main acute O2-sensing organ is the carotid body (CB), which contains neurosecretory chemoreceptor (glomus) cells innervated by sensory fibers whose activation by hypoxia elicits hyperventilation and increased cardiac output. Glomus cells have mitochondria with specialized metabolic and electron transport chain (ETC) properties. Reduced mitochondrial complex (MC) IV activity by hypoxia leads to production of signaling molecules (NADH and reactive O2 species) in MCI and MCIII that modulate membrane ion channel activity. We studied mice with conditional genetic ablation of MCIII that disrupts the ETC in the CB and other catecholaminergic tissues. Glomus cells survived MCIII dysfunction but showed selective abolition of responsiveness to hypoxia (increased [Ca2+] and transmitter release) with normal responses to other stimuli. Mitochondrial hypoxic NADH and reactive O2 species signals were also suppressed. MCIII-deficient mice exhibited strong inhibition of the hypoxic ventilatory response and altered acclimatization to sustained hypoxia. These data indicate that a functional ETC, with coupling between MCI and MCIV, is required for acute O2 sensing. O2 regulation of breathing results from the integrated action of mitochondrial ETC complexes in arterial chemoreceptors.

Neurobiology of the carotid body.

The carotid body (CB) is a bilateral arterial chemoreceptor located in the carotid artery bifurcation with an essential role in cardiorespiratory homeostasis. It is composed of highly perfused cell clusters, or glomeruli, innervated by sensory fibers. Glomus cells, the most abundant in each glomerulus, are neuron-like multimodal sensory elements able to detect and integrate changes in several physical and chemical parameters of the blood, in particular O2 tension, CO2 and pH, as well as glucose, lactate, or blood flow. Activation of glomus cells (e.g., during hypoxia or hypercapnia) stimulates the afferent fibers which impinge on brainstem neurons to elicit rapid compensatory responses (hyperventilation and sympathetic activation). This chapter presents an updated view of the structural organization of the CB and the mechanisms underlying the chemosensory responses of glomus cells, with special emphasis on the molecular processes responsible for acute O2 sensing. The properties of the glomus cell-sensory fiber synapse as well as the organization of CB output are discussed. The chapter includes the description of recently discovered CB stem cells and progenitor cells, and their role in CB growth during acclimatization to hypoxemia. Finally, the participation of the CB in the mechanisms of disease is briefly discussed.

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.

Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

Reduced expression of mitochondrial complex I subunit Ndufs2 does not impact healthspan in mice.

Aging in mammals leads to reduction in genes encoding the 45-subunit mitochondrial electron transport chain complex I. It has been hypothesized that normal aging and age-related diseases such as Parkinson's disease are in part due to modest decrease in expression of mitochondrial complex I subunits. By contrast, diminishing expression of mitochondrial complex I genes in lower organisms increases lifespan. Furthermore, metformin, a putative complex I inhibitor, increases healthspan in mice and humans. In the present study, we investigated whether loss of one allele of Ndufs2, the catalytic subunit of mitochondrial complex I, impacts healthspan and lifespan in mice. Our results indicate that Ndufs2 hemizygous mice (Ndufs2+/-) show no overt impairment in aging-related motor function, learning, tissue histology, organismal metabolism, or sensitivity to metformin in a C57BL6/J background. Despite a significant reduction of Ndufs2 mRNA, the mice do not demonstrate a significant decrease in complex I function. However, there are detectable transcriptomic changes in individual cell types and tissues due to loss of one allele of Ndufs2. Our data indicate that a 50% decline in mRNA of the core mitochondrial complex I subunit Ndufs2 is neither beneficial nor detrimental to healthspan.

Mitochondrial Redox Signaling in O2-Sensing Chemoreceptor Cells.

Significance: Acute responses to hypoxia are essential for the survival of mammals. The carotid body (CB), the main arterial chemoreceptor, contains glomus cells with oxygen (O2)-sensitive K+ channels, which are inhibited during hypoxia to trigger adaptive cardiorespiratory reflexes. Recent Advances: In this review, recent advances in molecular mechanisms of acute O2 sensing in CB glomus cells are discussed, with a special focus on the signaling role of mitochondria through regulating cellular redox status. These advances have been achieved thanks to the use of genetically engineered redox-sensitive green fluorescent protein (roGFP) probes, which allowed us to monitor rapid changes in ROS production in real time in different subcellular compartments during hypoxia. This methodology was used in combination with conditional knockout mice models, pharmacological approaches, and transcriptomic studies. We have proposed a mitochondria-to-membrane signaling model of acute O2 sensing in which H2O2 released in the mitochondrial intermembrane space serves as a signaling molecule to inhibit K+ channels on the plasma membrane. Critical Issues: Changes in mitochondrial reactive oxygen species (ROS) production during acute hypoxia are highly compartmentalized in the submitochondrial regions. The use of redox-sensitive probes targeted to specific compartments is essential to fully understand the role of mitochondrial ROS in acute O2 sensing. Future Directions: Further studies are needed to specify the ROS and to characterize the target(s) of ROS in chemoreceptor cells during acute hypoxia. These data may also contribute to a more complete understanding of the implication of ROS in acute responses to hypoxia in O2-sensing cells in other organs. Antioxid. Redox Signal. 37, 274-289.

Mitochondrial acute oxygen sensing and signaling.

Oxygen (O2) is essential for life and therefore the supply of sufficient O2 to the tissues is a major physiological challenge. In mammals, a deficit of O2 (hypoxia) triggers rapid cardiorespiratory reflexes (e.g. hyperventilation and increased heart output) that within a few seconds increase the uptake of O2 by the lungs and its distribution throughout the body. The prototypical acute O2-sensing organ is the carotid body (CB), which contains sensory glomus cells expressing O2-regulated ion channels. In response to hypoxia, glomus cells depolarize and release transmitters which activate afferent fibers terminating at the brainstem respiratory and autonomic centers. In this review, we summarize the basic properties of CB chemoreceptor cells and the essential role played by their specialized mitochondria in acute O2 sensing and signaling. We focus on recent data supporting a "mitochondria-to-membrane signaling" model of CB chemosensory transduction. The possibility that the differential expression of specific subunit isoforms and enzymes could allow mitochondria to play a generalized adaptive O2-sensing and signaling role in a wide variety of cells is also discussed.

Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

Lactate sensing mechanisms in arterial chemoreceptor cells.

Classically considered a by-product of anaerobic metabolism, lactate is now viewed as a fundamental fuel for oxidative phosphorylation in mitochondria, and preferred over glucose by many tissues. Lactate is also a signaling molecule of increasing medical relevance. Lactate levels in the blood can increase in both normal and pathophysiological conditions (e.g., hypoxia, physical exercise, or sepsis), however the manner by which these changes are sensed and induce adaptive responses is unknown. Here we show that the carotid body (CB) is essential for lactate homeostasis and that CB glomus cells, the main oxygen sensing arterial chemoreceptors, are also lactate sensors. Lactate is transported into glomus cells, leading to a rapid increase in the cytosolic NADH/NAD+ ratio. This in turn activates membrane cation channels, leading to cell depolarization, action potential firing, and Ca2+ influx. Lactate also decreases intracellular pH and increases mitochondrial reactive oxygen species production, which further activates glomus cells. Lactate and hypoxia, although sensed by separate mechanisms, share the same final signaling pathway and jointly activate glomus cells to potentiate compensatory cardiorespiratory reflexes.

Using redox-sensitive fluorescent probes to record real-time reactive oxygen species production in cells from mouse carotid body slices.

Reactive oxygen species (ROS) are important signaling molecules for physiologic processes such as acute response to hypoxia. However, reliable real-time ROS measurement in cells has been a long-standing methodological challenge. Here, we present a protocol to record acute changes in ROS production in sensory cells from mouse carotid body (CB) slices using redox-sensitive green fluorescent protein probes and microfluorimetry. This protocol provides sensitive and reproducible quantification of ROS during acute hypoxia in different subcellular compartments of CB glomus cells. For complete details on the use and execution of this protocol, please refer to Fernández-Agüera et al. (2015) and Arias-Mayenco et al. (2018).