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- Taste receptors in the Choroid Plexus are functional and regulated by sex hormonesPublication . Tomás, Joana Filipa Melfe; Gonçalves, Isabel Maria Theriaga Mendes Varanda; Santos, Cecília Reis Alves dosThe choroid plexuses (CPs) are highly vascularized structures constituted by a single layer of epithelial cells that project into the brain ventricles. The CPs are the main site of cerebrospinal fluid (CSF) production and constitute the Blood-CSF Barrier (BCSFB), holding high relevance in the surveillance of the CSF chemical composition. These structures contribute for the synthesis of biological compounds essential for the functioning and protection of the central nervous system (CNS) against neurotoxic insults. The expression of taste signalling pathway components in the CPs and its regulation by sex hormones was previously determined in a cDNA microarray study previously performed by our group. Moreover, the taste signalling pathway was determined as one as the top five pathways regulated by female sex hormones. The ectopic expression of sweet, umami and bitter taste signalling in extra oral organs have been extensively studied. In these organs, the taste receptors seems to behave as sensors to assess the composition of body fluids. The expression of the taste molecular machinery and its putative regulation by sex hormones in the CP raised the hypothesis that the taste signalling pathway could be one of the mechanisms involved in the monitoring of the chemical composition of blood and CSF at the BCSFB that my differ with gender. Considering this, we aimed to evaluate the presence and the functionality of the taste signalling pathway, as well as its regulation by the female sex hormones 17β-estradiol (E2) and progesterone (P4) in rat CP. In the first study, the presence and functionality of the taste signalling pathway was assessed. Transcripts for the taste-related genes Tas1r1, Tas1r2, Tas1r3, Tas2r109, Tas2r144, Gustducin, Plcb2, Ip3R3 and TrpM5 were found in CPs from adult Wistar rats. The expression of Tas1r1, Tas1r2, Tas2r144, Gustducin, Plcb2 and TrpM5 proteins was confirmed by Western blot, immunohistochemistry and immunocytochemistry. As umami and sweet receptors are heterodimeric receptors, we performed double labelling immunofluorescence, that showed the co-expression of T1R1 and T1R3 proteins that form the umami receptor, as well as, the coexpression of T1R2 and T1R3 proteins that form the sweet receptor. Having established the cellular localization of the taste machinery in CP epithelial cells (CPEC) we further evaluated the subcellular expression of taste proteins. For that, CPs were double labelled with antibodies for each of the taste-related proteins studied and a fluorescent marker of glycosylated surfaceexpressed proteins, revealing that taste-related proteins are located in the plasma membrane. After confirming the presence of the taste pathway molecular machinery, we proceed with functional assays. Considering that most of toxic/noxious compounds are bitter compounds that may exist in the CSF, we turned our attention to the bitter taste signalling pathway. Thus, to evaluate the functionality of the bitter pathway in primary cultures of CPEC we performed single cell calcium imaging assays using D-Salicin as the bitter stimulus. We observed an increase in intracellular Ca2+ evoked by D-Salicin that was diminished in the presence of the bitter taste receptors (T2Rs) blocker Probenecid, suggesting that T2R in the CPs are capable of sensing bitter compounds in the CSF and/or blood. An analysis in silico of our previous cDNA microarray data revealed that the decline of hormone levels in female rats upon ovariectomy clearly induced an up-regulation of the T2Rs Tas2r109, Tas2r124, Tas2r134, and Tas2r144, and the downstream effector molecules Plcb2 and Trpm5. Moreover, Tas2r109 and Tas2r144 were differentially expressed between female and male, showing a higher expression in males. This data led us to the second study of these thesis where the regulation of the taste pathway by female sex hormones was analyzed. For that, we compared the expression of taste-related genes in the CPs of sham and ovariectomized female Wistar rats and in CPs explants from newborn rats incubated with different concentrations of E2 and/or P4. Our results confirmed the cDNA microarray data, corroborating the regulation of taste-related genes by E2 and P4. The bitter receptors Tas2r109, Tas2r144, and the tasterelated genes Plcb2 and Trpm5 were down-regulated by ovarian hormones both in vivo and ex vivo. Functional implications of female sex hormones regulation was assessed, by single cell Ca2+ imaging, with the bitter compound, Denatonium Benzoate (DB), which is a known ligand of Tas2r144. Single cell Ca2+ imaging was performed in the immortalized CP epithelial cell line Z310 incubated with E2 and/or P4 in the presence of the respective hormone receptor blocker (fulvestrant or mifepristone, respectively). Intracellular Ca2+ variation, observed by single cell calcium imaging, was diminished in the presence of female sex hormones. However, while E2 effects were mediated via the nuclear E2 receptor, P4 effects were not abolished by the blocker of nuclear P4 receptor. Knocking-down Tas2r144 with a specific siRNA effectively reduced the Ca2+ response to the bitter compound DB, in a similar manner to E2 and P4, suggesting that female sex hormones down-regulated the responses of CPEC to chemical stimuli by reducing Tas2r144. In summary, our results confirmed and characterized the presence and functionality of the taste signalling machinery in CPs showing its regulation by female sex hormones. These results suggest that the taste signalling pathway may be one of the mechanisms by which the CP surveys the chemical composition of the CSF and elicit responses to modulate and maintain brain homeostasis. The achievements reached with this work will contribute to a better understanding of the mechanisms underlying the sensor/protective role of CPs in the CNS.