In order to characterize the role of leptin signaling and leptin receptors (LepR) in leptin transcytosis in tanycytes and to determine the role of chromogranins in the trafficking of leptin-containing vesicles in tanycytes, the fellow performed cell-sorting experiments using tdTomato reporter mice in which the TAT-Cre fusion protein was infused into the 3v, to selectively targets tanycytes (Annex-Fig.1A-B). Our data showed that purified tanycytes express transcripts for the vesicle-associated membrane proteins VAMP-2, -4 and -7. In addition, tanycytes express different forms of LepR, cubilin and its co-receptor megalin (Annex-Fig.1C-D). Data showed an increase in LepRb transcript in tanycytes after a fasting for 24h, thus suggesting changes in tanycytic receptors according to energy status (Annex-Fig.1E). These results report the functional importance of LepR in leptin signaling and transcytosis in tanycytes. The characterization of the transcytotic route taken by leptin is carrying out in collaboration with the laboratory of the Dr. Stéphane Gasman (CNRS UPR3212, Strasbourg), an expert in intracellular vesicular trafficking. Using specific markers for different intracellular compartments, internalized fluorescent-leptin is followed at different time points of the transcytotic process and leptin-containing compartments can be automatically detected allowing us to calculate their density and distribution and providing a complete road map of the route taken by leptin to reach the apical pole of tanycytes. We aimed also to characterize the role of SNARE in the exocytosis of leptin-containing secretory granules in tanycytes. To block SNARE-dependent exocytosis in tanycytes, the fellow used iBot mice, a transgenic mouse model in which protein VAMP2 is cleaved, and crossed them with the tdTomato line. Animals were injected to induce BoNT/B and Tomato expression in tanycytes and food intake was monitored. Data showed that selective inhibition of SNARE-dependent exocytosis in tanycytes caused differences in food intake (Annex-Fig.2A). A comprehensive analysis of the metabolism using metabolic cages showed differences between the two groups (Annex-Fig.2B-D). These results report the functional importance SNARE complex in the control of leptin trafficking and release in tanycytes. In addition, we are currently studying the involvement of VAMP 1-3 in leptin release in tanycytes expressing leptin coupled to the pH sensitive-GFP, and treated or not with TAT-Cre in the culture medium (Annex-Fig.3). The analysis of leptin levels released by iBot tanycytes will report the importance of VAMP-1-3 in leptin exocytosis. Besides, the fellow investigated the role of miRNAs in the leptin transport in tanycytes. Mice in which Dicer, an essential enzyme for miRNA biogenesis, was selectively inactivated in tanycytes were crossed with tdTomato mice. They were injected to induce Tomato expression and to inactivate Dicer in tanycytes. Data showed that deletion of Dicer in tanycytes caused a decrease of body weight (BW) and food intake (Annex-Fig. 4A-B). Also, this inactivation affect to feeding behaviour and impaired the glucose metabolism (Annex-Fig.4C-D). Moreover, in animals fed with high fat diet for 6 weeks, Dicer inactivation caused an increase in the food intake but not in BW (Annex-Fig.5A-B). Besides, using tanycytes from fed and 24 hours-fasted tdTomato and Dicer; tdTomato mice, the fellow is currently studying which miRNAs are involved in the in the transport of leptin in tanycytes. The exploitation and dissemination of the results has taken place as planned in the original proposal. All the results as well as the new methodology used in the research project was shared with the scientific community through: (i) multidisciplinary seminars and weekly meetings organized by the host group; (ii) International scientific conferences; and it will be also shared through the publication of the results in international peer-review journals with high imp
