Characterisation of the role of NG2-glia and microglia in hypothalamo-pituitary axis coupling.

80% of children affected by cancer now survive, 5 years after detection of the disease, thanks to earlier diagnoses and better treatments. This is a great improvement. However, these children require extra levels of monitoring and care because they can be affected by sequelae of anti-cancer treatments. In particular, brain radiation therapies are associated with a lifelong risk to develop pituitary hormone deficiencies. This risk is high, as up to 50% of survivors present these deficiencies. The pituitary is a small gland located at the base of the brain. It is attached to the hypothalamus, the region in the brain that controls the secretion in the blood of the six hormones the pituitary synthetizes: Growth Hormone, necessary for growth, Prolactin that induces milk production, Thyroid Stimulating Hormone inducing thyroid hormones levels to increase, Adrenocorticotropic Hormone that regulates secretion of the stress hormone cortisol, Luteinizing and Follicle-stimulating hormones both regulating puberty and reproduction. Because of the diverse roles of the pituitary hormones, deficits are associated with significant adverse effects including thyroid hormone and cortisol deficiencies, puberty defects, growth delay and cognitive impairments. In adults, pituitary deficits are also observed after treatment by radiotherapy for brain tumours, although the clinical effects are less profound. We know that radiation mostly affect the dividing cells, which is why they are used in anti-cancer therapy; however, we do not know how they induce a decrease in pituitary hormone levels. Understanding how this happens would help finding preventive measures or cures to avoid these deficits in paediatric cancer survivors. In animal models such as rats, cranial irradiation is also associated with pituitary hormones deficits, so rodents are a good model to study this phenomenon. In mice, as in humans, radiation therapies are also linked with a risk to develop obesity. Recently, scientists have demonstrated in mice that if radiations are focalised to the region that links the pituitary to the hypothalamus, obesity develops. They found that, in this small domain, hypothalamic nerves that regulate appetite are not sensing gut signals anymore. This is because the cells that protect them, called NG2 glia, divide frequently and are destroyed by radiations. In our lab, we have discovered that mice in which the Sox3 gene has been removed develop pituitary hormone deficits, as human patients carrying mutations in SOX3. Strikingly, we observe that NG2-glia is affected in Sox3 mutants, in this same region that links the hypothalamus and pituitary. The immune system may play a role, because when we give aspirin, likely to affect the inflammatory response in this context, normal hormone levels are restored in Sox3 mutants. We do not know whether aspirin has a similar effect in patients carrying SOX3 mutations. The goal of our project is to investigate the role of NG2 glia and brain immune cells in the communication between the hypothalamus and pituitary. We want to investigate if destruction of these cells by radiations explain the risk of irradiated cancer patients to develop pituitary deficiencies. To test our hypotheses, we will specifically eliminate the NG2 glia in mice using a novel approach and measure pituitary hormones. Little is known about the NG2 glia in this region: we will therefore examine in details which cells the NG2 glia is in contact with to understand better what its function could be. In parallel, we will test the potential involvement of brain immune cells by ablating them with a known efficient and selective drug after irradiation, and in Sox3 mutants, to examine whether hormonal deficits still develop in their absence. Finally, we will investigate the effects of aspirin in mice to understand how it can cure mice carrying Sox3 mutations. We hope that ultimately our results will help preventing pituitary deficits in cancer survivors.

The 5-year survival rate for paediatric cancers has increased to 80%. While extremely positive, this also means that sequelae of anti-cancer treatments become more prominent, such as pituitary hormone deficiencies found in up to 50% of survivors treated by brain irradiation. These are associated with significant morbidity, require monitoring, and replacement or substitution therapies. We do not know why these deficits develop, however cranial irradiation in rats is also associated with pituitary deficits so rodents are a good model to study their aetiology. Obesity can also develop following cranial radiotherapy. In mice, irradiation of the median eminence (ME), the structure at the base of the third ventricle linking hypothalamus and pituitary, drives this effect. More precisely, destruction of the proliferative ME NG2-glia leads to degeneration of Leptin Receptor positive dendrites that it normally supports, therefore loss of hypothalamic leptin sensing and consequently weight gain. We have moreover found that ME NG2 glia are affected in Sox3Y/- mutant mice which show hypopituitarism, as do patients carrying SOX3 mutations. Therefore, we hypothesize that destruction of ME NG2-glia by radiation therapy results in development of hypopituitarism in cancer survivors. To test our hypothesis, we will develop a novel model based on a brain sparing Diphteria Toxin to exclusively eliminate ME NG2-glia, as these are located outside the blood brain barrier. Moreover, microglia will be examined because aspirin can rescue Sox3Y/- hypopituitarism, suggesting an inflammatory contribution to the phenotype, which may also following irradiation. To investigate this aspect, we will eliminate the microglia pharmacologically in these contexts. Single cell analysis will be used to further characterize Sox3Y/- ME phenotypes. Our goal is to understand how hypopituitarism develops following radiation therapies to ultimately provide preventive measures or cures for cancer survivors.

Due to the substantial improvement in cancer diagnoses and treatments, the number of cancer patients surviving these diseases keeps increasing. Therefore, sequelae of anti-cancer treatments, such as deficiencies in pituitary hormones observed after brain radiation therapies, are likely to become more frequent. For this reason, it is important to understand their aetiology, as we propose here, and ultimately recommend preventive measures or cures to restore the best possible quality of life post-disease. It is especially important to prevent the occurrence of pituitary deficiencies in children as these can have life-long consequences, because lack of hormone(s) can cause cognitive deficits, delays or abnormal puberty, fertility problems, impairment of growth, and generally reduced quality of life. Our findings will be primarily beneficial and of use to the scientific community. Scientists expert in endocrinology, neurosciences, oncology and radiology are likely to represent key users of our results. In addition, we aim to develop a novel strategy where we will ablate brain specific cell types, exclusively in regions where the blood brain barrier does not exist. Technically it will be an asset to be able to implement this model, and beyond this project, to adapt it for other biological questions. Our project has a significant potential to be of use to the clinical community, and ultimately influence current treatment protocols, with the aim to avoid hypopituitarism following brain radiation therapies. Finally, because we will explore the use of specific compounds to ameliorate phenotypes, it is likely that pharmaceutical industry will be interested in the longer term.