Connexin 26-mediated breathing control by the healthy and obese brain

Humans produce about 1 kg of CO2 every day. CO2 dissolves in blood and will cause fatal changes in blood acidity unless its levels are precisely controlled. CO2-sensitive cells, present in the brain, control the depth of breathing to precisely regulate the level of CO2 in the blood and body.
Many human illnesses seriously affect to regulate breathing in response to levels of CO2. One of these conditions is obesity. 40% of obese patients have Obesity Hypoventilation Syndrome. These patients cannot breathe deeply enough to expel sufficient CO2, and have impaired CO2-sensitive regulation of breathing. Their blood is therefore more acidic, they feel sleepy, drowsy during daytime, exhaust easily and they have impaired sleep patterns, which result in reduced ability to work, a vicious cycle. Other human conditions, for example Chiari Syndrome, which affects around 1% of people in middle age, leads to severe debilitating problems, ranging from headaches to inabilities to swallow, sleep and breathe, patients suffer from apneas, shallow and irregular breathing during sleep at night.
We have recently discovered key cells responsible for sensing of CO2 on the brain's surface, which sheds unexpected new light onto the fundamental question how everybody's brain senses CO2 and provides new avenues to understand the medical conditions mentioned above. An essential molecule inside these cells, a membrane channel called Connexin26, helps these cells to sense CO2 levels. When only a small group of specific cells, no more than 25, are made to lose the Connexin26 gene by a novel genetic method, the ability of the body to regulate breathing in response to higher CO2 levels (comparable to the CO2 levels inside someone's breath) is reduced by 40%. These 25 critical cells develop around puberty in rodents, are retained into late middle age and then lost in old age, thus act in a specific time window during life.
Connexin 26 is also active in other cells around the brainstem, however their function in relation to breathing is unknown. We propose to inactivate this gene in further specific groups of cells in transgenic rodents to test whether and how these cells regulate the sensitivity of breathing to CO2. We have discovered that these CO2-sensing cells carry receptors for an important pathway that controls body weight (the leptin pathway). In obese patients, the leptin pathway is impaired. This means that we may have found a missing link between the mechanisms underlying obesity, and the defective regulation of breathing in these patients: the CO2-sensing cells may not be able to sense CO2 well enough as a consequence of impaired leptin signalling. As we can now observe and characterize the properties of these very important cells in animals directly, we can ask whether obesity, induced by high fat/calorie diet during young age, affects the development and properties of these critical CO2-sensory cells. We propose to test further this hypothesis experimentally by altering leptin signalling specifically in the CO2-sensing cells that we have discovered, to see whether we can recapitulate the human Obesity Hypoventilation Syndrome in rodents. With such animal model one can develop and test new drugs that help patients breathe. Our work bears the promise of great relevance to humans. Do those important sensor cells die if we eat too much sugar during adolescence? How are they affected, is that permanent? Can it be reversed by diet or medicines? Which genes do they turn on? If we know how these essential CO2 sensory cells are affected by diet, we can be much more precise on recommending the right types of food to be eaten during adolescence and puberty in humans. Breathing is essential for all we do, whether we are awake or sleep and obesity is a growing global health epidemic. By linking developmental genetics and physiology this project promises to provide long-term benefit to the physical and mental well-being of the wider community.

Expiration of the CO2 we produce is essential for survival. This is centrally controlled in the medulla. We have shown that CO2 is sensed directly by the brain: It gates the opening of Connexin26, leading to ATP release and increase in breathing volume. We ablated Cx26 from specific tissues of the medulla using recombinase mediated gene ablation. To study mutant breathing responses to hypercapnia we established ATP release assays on medullar slices and whole-body plethysmography in awake animals. We discovered a novel Cx26+ chemosensory cell type derived from the neural crest, which develops in middle age and dies in old age. Ablation of Cx26 inside these cells reduces breathing volume by 40% upon mild hypercapnia. We now wish to learn how epigenesis affects the development of and signalling inside these cells. We will also explore other areas of Cx26's function in central control of respiration: endothelia, leptomeninges and the rostral chemosensing region. Recombinase-mediated conditional gene ablation and lineage labelling, using Cre lines under cell- or region specific control and floxed Cx26 or LeprB alleles. Mutant phenotypes will be assayed as mentioned. As chemosensory cells carry the leptin receptor, a possible powerful modulator of breathing, they might be defective in obesity hypoventilation syndrome. By ablating the leptin-receptor inside these cells we will test how leptin affects their ontogeny and/or function. As chemoreceptors are now genetically tractable we will ablate Cx26 and LepRB inside them & probe transcriptomes at single cell resolution in relation to diets, yielding new pathways and drugs targets. We establish respiration-specific animal models for human conditions with blunted chemosensitivity: Chiari, SIDS, CCHS all leading to serious morbidity or mortality. Understanding these now tractable chemosensors will enable stratification of patient populations and help meet the challenges of an ageing and increasingly obese patient population.

Pathway to Impact summary.
Our project aims at harnessing the power of genetic analysis to start to tackle a very important biomedical problem: How exactly the brain senses the levels of CO2 and uses this to regulate the depth of breathing. In question 1 we study the role of Cx26 in the vascular system upon central breathing control, in question 2 we investigate its role upon one central respiratory generator, in question 3 we see whether and how the cells that sense CO2 are directly affected by the leptin pathway which is central to body energy control and misregulated in obesity. In question 4 we look whether and how diet during adolescence affects the development of these chemosensory cells which we have now rendered tractable and visible for the first time.

The outcomes of this research are likely to affect the way how we think about the complex interplay between epigenetic,external, nutritional factors and internal signalling events within the central nervous systems that affect deep breathing in response to elevated CO2. As the cells doing the sensing are likely to respond to leptin signalling and might change their behaviours as a result of this. Our results might also affect the way we think about nutrition in childhood/adolescence and its long-term consequences for breathing during adulthood. Studying leptin signalling and breathing at the mechanistic level affects society in general with an ever growing obesity epidemic (due to dietary mis-regulation of the leptin pathway that is modifiable by new cultural practices).

It affects patients in particular who suffer of the consequences of breathlessness, shortness of breath in many ways: this leads to problems during sleep - severely denting its recuperating effect and it has societal impact because the inability of obese patients to react upon higher levels of acidity in their blood by deeper breathing - makes them sleepy and less able to cope with the challenges of life, leading to reduced economic performance. Moreover, doctors having to treat obese patients in intensive care, respiratory (and emergency) medicine need to deal with obesity hypoventilation syndrome on a regular basis. In our impact strategy we therefore wish to target the general public (via Youtube clips), medical and health professionals (via direct engagement, talks, common conference activities), charities of affected patients as well as companies developing novel drugs against obesity. We wish to target both the UK and US health sector via existing links and develop these further in the course of our proposed 4 year project. (see Pathway to Impact statement for further details on our strategy).