Tanycytes shuttle leptin into the metabolic brain: mechanistic insights and role in the pathophysiology of hormonal resistance and diabesity – GlioShuttles4Metabolism

The premise of ‘GlioShuttles4Metabolism’ is that the design of successful treatments for obesity and associated disorders will depend on an improved understanding of how circulating metabolic signals enter the brain to trigger homeostatic responses. In addition, we believe that genuine progress in developing effective preventive and therapeutic strategies to tackle obesity and associated metabolic diseases requires novel ideas and a multifaceted approach with state-of-the-art technologies, only possible with an interdisciplinary consortium of basic scientists committed to the same goal as a single functional unit.
Partner 1, in association with Partner 2, will delineate the importance of leptin receptors and co-receptors in leptin transcytosis in primary cultures of tanycytes and brain slices, and determine the effect of ER stress on leptin transport and signaling in vitro. Partner 1 will use animal models to validate the results obtained in vitro and determine the functional consequences of the selective invalidation of leptin receptors and co-receptors in tanycytes on metabolic regulation in mice. Partner 3 will interact closely with Partners 1 and 2 to explore the role of activity-dependent exocytosis in the release of the endocytosed leptin at the apical pole of tanycytes, a process that is hampered in DIO and is thus a potential therapeutic target for the treatment of central hormonal resistance. Finally, Partners 1 and 2 will interact to engineer an in vitro model system of the hypothalamic blood-CSF barrier that promises to be an extremely sensitive tool for the study of the passage of blood-borne metabolic signals into the brain both under physiological and pathological conditions.

To visualize leptin transport into tanycytes, median eminence tanycytes have been locally puffed at the level of their endfeet with a far-red fluorescent bioactive leptin. Unexpectedly, however, the puffs of fluorescent leptin did not allow its capture by tanycytic endfeet and processes in the living brain slices; they caused important tissue remodeling and the fluorescent leptin spilled over and quickly gained access to the median eminence tissue.
Using an alterative approach, we have been able to demonstrate that tanycytes responded to leptin and that this response triggered calcium waves in tanycytes in living brain slices obtained from mice expressing the GCamp3 protein under the promoter of TPRM5, which is selectively expressed in tanycytes (see figure bellow). Importantly, performing bath-perfusion of the point-mutated leptin LAN before selectively puffing leptin onto tanycytes abrogated this response.

This proposal requires extensive knowledge and methodological expertise in the neurobiology of obesity and diabetes, physiology, biochemistry, molecular and cellular biology and animal models, not readily available in a single laboratory setting. Because we have assembled a knowledgeable team of scientists who have developed the necessary expertise in each of the proposed experimental set-ups, we are confident that we can achieve the challenging goals of the “GlioShuttles4Metabolism” project.

• Conférences:

1. Prevot V. «Tanycyte transport of leptin into the hypothalamus: Implications in leptin resistance«, 55th Annual Meeting of the European Society for Paediatric Endocrinology (ESPE), Symposium. Paris, France, September 2016.
2. Prevot V. «CSF Barriers in Neuroinflamation«, Neurosciences in Intensive Care International Symposium 2016 (NICIS2016), Symposium. Institut Pasteur, Paris, France, June 2016.

• Communications orales:

1. Duquenne M, Caron E, Clasadonte J, Pfrieger FW, Prevot V. A role for tanycyte exocytosis in the central control of energy homeostasis? Keystone Symposia: Neuronal Control of Appetite, Metabolism and Weight, Workshop, Copenhagen, Denmark, may 2017
2. Duquenne M, Caron E, Pfrieger FW, Prevot V. A role for tanycyte exocytosis in the central control of energy homeostasis? 42ième Colloque de la Société de Neuroendocrinologie. Oral communication, Dijon, September 2017 (Best oral presentation award).

• Communications affichées:

1. Millet M, Manon D, Haeberlé A-M, Prevot V, Ory S, Gasman S. Molecular mechanisms of leptin transport accross the tanycyte of the median eminence. 42ième Colloque de la Société de Neuroendocrinologie. Poster, Dijon, September 2017.

Obesity and its metabolic consequences (type-2 diabetes, cardiovascular, gastrointestinal and reproductive disorders, as well as certain cancers) have become major causes of morbidity and mortality in developed countries. In France today, 50% of adults (22 million) are overweight, of which 7 million are obese, and the past 30 years have witnessed an alarming 7-fold increase in the incidence of childhood obesity. The development of effective preventive and therapeutic measures for these disorders and the reduction of the associated medical, familial and socioeconomic burdens is impossible without an improved understanding of the pathways and mechanisms leading to obesity. Among these mechanisms is the transport of peripheral metabolic hormones into the brain, a critical limiting step whose deregulation leads to these disorders.
Current evidence indicates that the brain is a key site processing information from “adiposity signals” such as the anorexigenic hormone leptin, which circulates in proportion to body fat mass and instructs the individual to stop feeding. Leptin receptors are expressed in regions of the hypothalamus involved in the control of food intake and energy homeostasis, and injections of leptin into the brain of leptin-deficient mice eliminate overfeeding. Conversely, the deletion of leptin receptors or the blockade of leptin entry into the brain results in obesity and resistance to this hormone.
During previous work supported by the ANR (GLIODIABESITY, 2009-2013), we raised the groundbreaking notion that tanycytes, a specific type of hypothalamic glial cells, act as “gatekeepers” that regulate the access of blood-borne signals to the hypothalamus (Langlet et al., Cell Metab 2013), and in particular, its vesicular transport into the cerebrospinal fluid, from where it enters other leptin-sensitive regions (Balland et al., Cell Metab 2014). We have shown that this tanycyte-mediated transport of leptin is suppressed in both genetic and diet-induced obesity (Balland et al., Cell Metab 2014). Reversing leptin resistance by restoring tanycytic leptin transport holds important therapeutic potential (Patent WO 2014141124 A1, PI: Prevot V), as shown by previous positive reviews by the ANR, as well as two Marie Sklodowska Curie postdoctoral fellowships in 2015 to pursue this work. However, the molecular mechanisms involved in the tanycytic shuttling of blood-borne leptin into the hypothalamus are still largely unknown.
The overall objective of this proposal is to further develop this highly original angle by developing state-of-the-art approaches to i) characterize leptin transporters and the trans-tanycytic route used by peripheral leptin to enter the metabolic brain (Aims 1 and 2), ii) assess whether endoplasmic reticulum stress, which is involved in leptin resistance, alters tanycytic leptin transport (Aim 3), and iii) develop novel models and cutting-edge tools to probe this tanycytic barrier in vitro (Aim 4). To carry out this innovative project in a highly competitive field of biomedical research, we are now teaming up with a group of internationally renowned experts in leptin signaling who have contributed to the aforementioned discoveries (Partner 2: Ralf Jockers & Julie Dam), and a second partner expert in intracellular vesicular trafficking (Partner 3: Stéphane Gasman).
We are convinced that the successful outcome of the proposed research plan will have important implications for public health by providing essential clues about how the peripheral hormone leptin, which carries metabolic information to the CNS both during development and adulthood, enters the brain. More broadly, this research promises to shed new light on the cellular and molecular mechanisms used by the hypothalamus to integrate endocrine signals that coordinate energy homeostasis. The results will pave the way for the development of new treatment strategies to overcome hormone resistance in human obesity and associated metabolic syndromes.