Taste receptors in the Choroid Plexus are functional and regulated by sex hormones

The 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.Os plexos coróides (PCs) são estruturas altamente vascularizadas localizadas nos ventrículos cerebrais. São constituídos por uma monocamada de células epiteliais cuboides assentes numa membrana basal que está em contacto direto com um estroma altamente vascularizado, com tecido conjuntivo rico em fibroblastos e com células do sistema imunitário. O lado apical destas estruturas está em contacto direto com o líquido cefalorraquidiano e apresenta microvilosidades e alguns cílios. O lado oposto, o lado basal, está em contacto com vasos sanguíneos altamente fenestrados. Os PCs formam vilosidades de modo a aumentar as superfícies de contacto entre as células epiteliais do lado apical com o líquido cefalorraquidiano, e com o fluído intersticial do lado basal. Os PCs são o principal local de síntese de líquido cefalorraquidiano, estão envolvidos na vigilância imunológica do sistema nervoso central, são estruturas ativas na neurogénese e são ainda responsáveis pela remoção de compostos tóxicos e metabolitos do líquido cefalorraquidiano resultantes de processos metabólicos do sistema nervoso central. A sua ultra estrutura é composta por células unidas através de junções de oclusão que permite que este órgão forme uma barreira entre o sangue e o líquido cefalorraquidiano, a barreira sanguelíquido cefalorraquidiano, que previne o movimento de substâncias para dentro e fora do cérebro pelos espaços intercelulares. Tendo em conta a sua estrutura e localização, os PCs têm um papel crucial na monotorização da composição química do líquido cefalorraquidiano e do sangue, contribuindo para a síntese e/ou transporte de compostos essenciais para um normal funcionamento e proteção do sistema nervoso central contra agentes neurotóxicos. A expressão de genes relacionados com a via de transdução de sinal do paladar no PC foi detetada num estudo de microarrays de DNA complementar, realizado previamente pelo nosso grupo de trabalho, em PCs de ratos castrados Wistar Han. Nesse estudo, verificou-se ainda que a via de sinalização do paladar foi uma das cinco vias mais afetadas pelas hormonas sexuais femininas, estando sobre expressa em ratos fêmea ovariectomizados. A expressão da via do paladar tem sido amplamente estudada, fora da cavidade oral, em órgãos como o estômago, intestino, pulmões, coração, testículos, artérias, entre outros. Nestes órgãos, os recetores do paladar parecem funcionar como sensores que monitorizam a composição química dos fluidos biológicos circundantes que, quando ativados, desencadeiam respostas metabólicas defensivas. A via de transdução de sinal do paladar, descrita originalmente nas células sensoriais nos botões gustativos, inicia-se com a ligação das moléculas do sabor ao respetivo recetor do paladar (Tas1r2/Tas1r3 e Tas1r1/Tas1r3, respetivamente para o doce e umami, e Tas2r para o amargo) ativando uma via de transdução de sinal que resulta na despolarização da célula. Os ensaios experimentais apresentados nesta tese tiveram como objetivo geral investigar o papel da via do paladar na capacidade de monitorização da composição química do líquido cefalorraquidiano e/ou sangue por parte dos PCs. Deste modo, o presente estudo teve como objetivos específicos a análise da expressão dos diferentes componentes da via de sinalização do paladar e o estudo da sua funcionalidade no PC de rato, bem como a avaliação da sua regulação pelas hormonas sexuais. No primeiro trabalho desenvolvido, avaliou-se a expressão e a funcionalidade da via do paladar no PC de rato, por RT-PCR e singel cell calcium imagingI, respectivamente. Identificaram-se transcritos de genes da via de sinalização do paladar tais como os recetores do paladar Tas1r1, Tas1r2, Tas1r3, que formam os recetores do doce e umami, os recetores Tas2r109 e Tas2r144 que detetam compostos amargos, e moléculas da maquinaria da via de sinalização (Gustducina, fosfolipase beta 2, inositol tri-fosfato e o membro 5 da subfamília M do canal recetor de catiões com potencial transitório). A expressão das respetivas proteínas foi confirmada por Western blot, imunofluorescência, imunohistoquímica e imunocitoquímica. Uma análise imunocitoquímica de explantes de PC com marcação dupla da proteína alvo (Tas1r3 e Tas2r144) e do marcador de células epiteliais de PC, a transtirretina, revelou que os recetores do paladar estão localizados nas células epiteliais de PC. Uma vez confirmada a expressão de toda a maquinaria da via de transdução de sinal do paladar no PC, procedemos aos estudos funcionais. Sabendo que a maioria dos compostos tóxicos e/ou nocivos correspondem a compostos amargos, selecionámos como alvo do nosso estudo os recetores do amargo, dado que a sua presença no PC poderá estar associada à deteção de compostos neurotóxicos. De modo a avaliar a sua funcionalidade, utilizámos culturas primárias de células epiteliais de PC onde realizámos a técnica de single cell calcium imaging utilizando a D-Salicina como composto amargo. O estímulo com D-Salicina provocou um aumento nos níveis intracelulares do ião cálcio que na presença de um bloqueador de recetores do amargo, o Probenecid, diminuíram de intensidade. Uma vez que a presença de um bloqueador dos recetores do amargo diminui a resposta observada, é muito provável que esta se deva à ativação dos recetores do amargo. Deste modo, podemos afirmar que os recetores do amargo presentes no PC são funcionais, podendo detetar compostos amargos presentes no líquido cefalorraquidiano e/ou no sangue. O segundo estudo desenvolvido, teve por base uma análise de dados de microarrays de DNA complementar de PC de ratos castrados, realizado previamente pelo nosso grupo. A análise in silico destes dados mostrou que o declínio das hormonas sexuais femininas aumentava significativamente os níveis de expressão dos recetores do amargo Tas2r109, Tas2r124, Tas2r134 e Tas2r144, e as moléculas da via de sinalização do paladar Plcb2 e Trpm5. Mostrou também que os recetores do amargo Tas2r109 e Tas2r144 são diferencialmente expressos entre machos e fêmeas, apresentando uma expressão mais elevada nos machos. Deste modo, no segundo estudo apresentado nesta tese, analisámos a regulação da via de transdução do paladar pelas hormonas sexuais femininas. Assim, estudámos a expressão dos genes Tas2r109, Tas2r144, Plcb2 e Trpm5 no PC de ratos fêmea castrados e não castrados e em explantes de PC de ratos recém-nascidos (5-6 dias de idade) incubados com diferentes concentrações de estradiol e progesterona. Os resultados obtidos, in vivo e ex vivo, corroboram os resultados dos microarrays de DNA complementar comprovando a regulação hormonal dos genes da via de transdução de sinal do paladar no PC. O efeito das hormonas sexuais na resposta das células do PC a um estímulo amargo foi avaliado por single cell calcium imaging com o composto Benzoato de Denatónio, um ligando do recetor do amargo Tas2r144, numa linha celular imortalizada de células epiteliais de PC (Z310) em presença de estradiol e/ou progesterona e também na presença dos bloqueadores dos recetores nucleares das hormonas. Observámos que a presença de estradiol e/ou progesterona diminuiu a amplitude de resposta das células Z310. O estudo com os bloqueadores hormonais permitiu-nos concluir que o efeito do estradiol parece ocorrer via recetor nuclear, e os resultados com progesterona sugerem o envolvimento dos recetores membranares da progesterona. Com o objetivo de analisar se as respostas observadas por single cell calcium imaging ocorrem via recetor Tas2r144, realizaram-se ainda ensaios de cálcio com o recetor Tas2r144 silenciado com um RNAi específico. Os ensaios de cálcio após silenciamento do Tas2r144 mostraram uma redução significativa da resposta ao Benzoato de Denatónio, de maneira semelhante aos efeitos do estradiol e progesterona, sugerindo que as hormonas sexuais femininas regulam negativamente as respostas do PC a estímulos químicos, reduzindo a Tas2r144. A regulação exercida pelas hormonas sexuais na resposta a compostos amargos no PC, eventualmente neurotóxicos, poderá estar envolvida na diferença no aparecimento e progressão de doenças do sistema nervoso central entre géneros. Em suma, os nossos resultados confirmam a presença da maquinaria molecular da via de transdução de sinal do paladar no PC de rato, mostrando que a via do amargo está funcional, e revelam a sua regulação pelas hormonas sexuais femininas. Os resultados obtidos neste trabalho suportam a hipótese de que a via de transdução do paladar poderá ser um dos mecanismos utilizado pelo PC para monitorizar o conteúdo químico do líquido cefalorraquidiano e/ou sangue e responder de modo a modular e manter a homeostasia do sistema nervoso central. No futuro, os resultados apresentados poderão contribuir para uma melhor compreensão dos mecanismos por detrás da função do PC em monitorizar/proteger o sistema nervoso central

Investigation Into The Molecular Mechanisms Underlying Idiopathic Intracranial Hypertension

Idiopathic intracranial hypertension (IIH) is a neurological disorder characterised by raised cerebrospinal fluid (CSF) pressure in the absence of any intracranial pathology. IIH mainly affects obese women between the ages of 15 and 45. Two possible mechanisms that could explain the increased CSF pressure in IIH are excessive CSF production by the choroid plexus epithelium or impaired CSF drainage back into the venous blood but the molecular mechanisms controlling these in IIH remain to be determined. In vivo ventriculo-cisternal perfusion and variable rate infusion techniques assessed changes in rates of CSF secretion/resistance to CSF drainage in male and female Wistar rats fed either a normal control or high-fat (HF) diet, following treatment with inflammatory mediators already found to be elevated in the CSF of IIH patients: chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-17 (IL-17), IL-6, IL-1β, tumour necrosis factor-α (TNF-α), as well as adipocyte-derived hormone leptin and the glucocorticoid hydrocortisone (HC). Female Wistar rats raised on a HF diet were shown to have the highest CSF secretion and lowest CSF drainage rates under untreated conditions. Increased CSF secretion was observed in rats of all genders and diets following TNF-α or HC treatment, however the greatest increase by TNF-α and HC over basal levels was observed in female rats raised on a HF diet. In addition, female rats on a HF diet, treated with CCL2 or IL-17, displayed an increase in resistance to CSF drainage when compared to untreated controls (indicating lower levels of CSF drainage). Therapies targeting HC, TNF-α, CCL2 and/or IL-17, whether separately or in combination, may be beneficial to modulate rates of CSF secretion and/or resistance to CSF drainage pathways, both factors likely contributing to the raised intracranial pressure observed in obese female IIH patients

Biomedical Perspectives II

Abstract book of International Scientific Conference of Students, Postgraduates and Young Scientist

Glucocorticoids

As one class of the most important steroid hormones, glucocorticoids have long been recognised and their therapeutic benefits have been widely used in clinical treatment, especially in anti-inflammation cases. Glucocorticoids regulate various processes in the body including the mobilization of energy stores, immune functions, gene expression, and maintenance of the homeostasis as well as the stress response, this is not surprising that the concept of "glucocorticoids" is mentioned in almost all medical text books that focus on specific organs or systems such as the cardiovascular system, the immune system, and the neuroendocrine system. The book of Glucocorticoids - New Recognition of Our Familiar Friend aims to introduce the latest findings relating to glucocorticoids, either freshly from the laboratory or from clinical case studies, and to open up a new angle of looking at the issue of balancing the therapeutic benefits and side effects brought up by glucocorticoids

Abstracts of the International Medical Students’ Congress of Bucharest (IMSCB) 2019

Abstracts of the International Medical Students’ Congress of Bucharest (IMSCB) 2019

Neuroendocrinology and Behavior

Understanding of the neuroendocrine system provides an insight into a wide range of bodily and mental processes. Neuroendocrinology and Behavior brings its readers a concise guide to up-to-date knowledge on the function of the endocrine glands and organs in association with neurotransmitters, neuropeptides, and behavioral manifestations. Various forms of stress response, e.g. anxiety and cognitive changes, have been intrinsic to both ourselves and other species. Specifically, these mechanisms involve complex interplays among physical, emotional, and behavioral processes in humans. This volume provides information on peptide hormones, i.e. oxytocin and vasopressin, and how studies of these neuropeptides enrich our understanding of social and other behaviors. These neuromodulators also affect sexual behavior and water balance in the body, an issue also addressed in this Book

Testicular morphological and biochemical perturbations in experimental animals under antiretroviral therapy and the role of Naringenin, a bioactive flavonoid.

Doctoral Degree. University of KwaZulu-Natal, Durban.Declining male fertility is one of the neglected concerns of people living with HIV/AIDS in spite of a dual outlook of a social and a health dilemma. This issue of infertility is particularly relevant as majority of affected individuals are in their reproductive years. This thesis examines the impacts of the Fixed Dose Combination (FDC) of Highly Active Antiretroviral Therapy (HAART) Tenofovir/ Emtricitabine and Efavirenz (TDF/FTC/EFV) on the male reproductive capacity. It also explores the protective potentials of a bioactive flavonoid, Naringenin in testicular perturbations. The study was motivated by two major research questions namely: (1) what are the impacts of the recently approved first line antiretroviral therapy for adults FDC, TDF/FTC/EFV on the testes? (2) What is the role of Naringenin in alleviating testicular perturbations induced by HAART? Previous studies point to the negative impacts of the older generation of FDC of HAART on the semen quality and histomorphometry of the testes following a long-term use. The study addresses both the long-term and short-term use of antiretroviral drugs as observed in pre-exposure prophylaxis (PrEP) and post exposure prophylaxis (PEP). The research assesses the impacts of the drugs on the reproductive capability as well. Findings from this study support the argument that the negative effects of the drugs were consequent upon both the short-term and long-term use. To illustrate this hypothesis, the study was conducted in two distinct phases. The first phase which lasted a total of 28 days considered the duration of PEP or PrEP. The second phase which lasted a total of ten weeks captured all the stages of spermatogenesis in rats. In this phase male Sprague Dawley rats were exposed to fertile females after the treatments. The study thus advances an understanding of the mechanism of HAART-induced testicular injury. A therapeutic dose of TDF/FTC/EFV adjusted for animal weight was aministered on a total of 48 animals randomly divided into 6 equal groups each with a different treatment as follows; Group A: Control (Distilled water); Group B: HAART (TDF/FTC/EFV), Group C: Naringenin, 40 mg/kg; Group D: Naringenin, 80 mg/kg; Group E: HAART + Naringenin, 40 mg/kg; Group F: HAART+ Naringenin, 80 mg/kg. At the end of each phase, harvested testes were subjected to histomorphometry and ultrastructural analysis. The caudal epididymis was assessed for semen parameters and sperm mitochondrial DNA (mtDNA) fragmentation. Biochemical parameters such as serum levels of reproductive hormones (Luteinizing hormone and Testosterone) and intratesticular antioxidant enzyme activities were assayed. Contrary to prior beliefs, this research reveals that the immediate effects following short-term use of HAART are far more deleterious. This finding is consequent upon the significant drop in the sperm count (p˂0.001) and sperms with normal morphology (p˂0.001) compared to (p˂0.01) in the long-term. Histomorphometric analysis also revealed a significantly shrunken seminiferous tubule following a short-term use. These outcomes were associated with an increase in the mtDNA fragmentation in group B when compared to control (p˂0.05). Naringenin reversed abnormalities in groups E and F, displaying better semen parameters in both count and motility. Serum levels of testosterone were altered in both phases. The overall effects of all these changes were observed in the pregnancy rate which reduced in group B when compared to all the other groups. This study established that HAART has deleterious effects on the testicular microanatomy and function. These effects may impact on steroidogenesis and ultimately spermatogenesis. It consequently impairs fertility while Naringenin promises to be a potential complimentary adjuvant especially in the short term therapies. Keywords: HAART, semen parameters, reproductive hormones, testicular ultrastructure, 3 beta hydroxysteroid dehydrogenase