Mechanisms Mediating Internalization and Transport of Ghrelin in Hypothalamic Tanycytes

GÓMEZ, IVANA; CASTROGIOVANNI, DANIEL; URIARTE, MAIA; PERELLO, MARIO; DE FRANCESCO, PABLO NICOLÁS

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biocencias; 2022

Sociedad Argentina de Investigación Clínica

Hypothalamic tanycytes are polarized glial cells that line the base of the third ventricle. Their somas contact the cerebrospinal fluid (CSF), while their terminal side (endfeet) contact the capillaries of the blood-brain barrier (BBB) or the fenestrated capillaries of the median eminence, forming a relevant anatomical interface for the transport of molecules between blood and CSF. Using mice and rats, we recently described that tanycytes internalize the orexigenic hormone ghrelin in vivo and in vitro. Here, we study the cellular mechanisms of ghrelin uptake by hypothalamic tanycytes and its transport direction. Specifically, we incubated primary cultures of rat hypothalamic tanycytes with a fluorescent variant of ghrelin (Fr-ghrelin) in basal conditions or after pharmacological blockage of either clathrin-mediated internalization using Pitstop, or intracellular transport using colchicine. We also coincubated tanycytes with Fr-ghrelin and native ghrelin. We then quantified fluorescence intensity in soma, process and endfeet of each cell. Data were compared using Kruskal Wallis test with Dunn’s post hoc test. We found that intracellular fluorescence: 1) is found predominantly in the soma of tanycytes after a 5 min incubation (K-W p<0.0001; p<0.0001); 2) increased in somas (K-W p<0.0001; p= 0.003), processes (K-W p<0.0001; p< 0.0001) and terminals (K-W p<0.0001; p< 0.0001) after 30 min incubation as compared to 5 min incubation; 3) was reduced only in terminals in the presence of colchicine (K-W p<0.0001; p< 0.0001); 4) was decreased in all compartments in the presence of Pitstop (K-W p<0.0001; p<0.0001); and 5) was reduced in somas (K-W p<0.0001; p< 0.0001), processes (K-W p<0.0001; p= 0.0018) and endfeet (K-W p<0.0001; p= 0.0027) after coincubation with native ghrelin. This evidence shows that tanycytes are able to specifically internalize ghrelin through clathrin-mediated endocytosis in the somas and presumably to transport it from the apical to the terminal side.