Loading...
2 results
Search Results
Now showing 1 - 2 of 2
- Characterization of bitter taste receptors expression and function in the human blood-cerebrospinal fluid barrierPublication . Duarte, Ana Catarina Abreu; Santos, Cecília Reis Alves dos; Gonçalves, Isabel Maria Theriaga Mendes VarandaBitter taste receptors (TR2) expression and functionality was recently reported in the rat choroid plexus (CP). CP epithelial cells establish a major brain barrier, the blood-cerebrospinal fluid barrier (BCSFB). Given their capacity to bind a large array of chemical compounds, we hypothesised that TR2 might be involved in monitoring the composition of blood and cerebrospinal fluid. Brain barriers play a critical role in the protection of the central nervous system (CNS) by hindering the access of toxic substances to the brain. Consequently, many drugs targeting neurological disorders are impaired to cross these barriers. This is explained through the expression of several membrane transporters in brain barriers cells that efflux drugs, thus impairing drug cell accumulation in the brain. A wide range of compounds that bind to TR2 show neuroprotective and anti-tumoral properties. However, their low bioavailability in the CNS restrains its therapeutic application. Additionally, bitter compounds might interact with transporters that are also found in brain barriers. Therefore, bitter compounds might be effluxed which explains their low bioavailability but can also regulate the action of these transporters in order to increase their or other drugs’ intracellular accumulation. Considering that bitter compounds are TR2 agonists it is possible that TR2 play an important role on the bioactive effects of bitter compounds in the CNS, as reported in other tissues. The main goal of this doctoral thesis was to analyse the expression and function of the bitter signalling pathway in the human BCSFB. Additionally, the role of human TR2 (TAS2Rs) as modulator of specific neuroactive bitter compounds on ABC transporters function and activity at the BCSFB was also evaluated. The first research work presented showed the expression of 13 TAS2Rs as well as of downstream effector proteins of the taste signalling pathway in the human BCSFB. Moreover, we demonstrated that TAS2R14 and TAS2R39 are functional in a human cell model of the BCSFB and respond to bitter compounds quercetin and chloramphenicol, respectively. The second research work evaluated resveratrol transport across the BCSFB and the involvement of TAS2Rs. Results showed that resveratrol is able to cross the BCSFB from blood to cerebrospinal fluid in a dependent manner of TAS2R14 expression at CP epithelial cells. Further, efflux transporters ABCC1, ABCC4 and ABCG2, which are expressed at CP epithelial cells, transport resveratrol. Additionally, resveratrol upregulated ABCG2 expression and regulated ABCC4 and ABCG2 efflux activity in TAS2R14 dependent way. In conclusion, the results obtained during this project demonstrate that TAS2Rs are expressed and functional at the human BCSFB and support their participation in the monitorization of chemical composition of the surrounding fluids. Furthermore, the major achievements of this thesis strongly support the role of BCSFB in the regulation of the transport of molecules into the brain. In the future, it is necessary to further exploit the role of other TAS2Rs as mediators of the effects of bitter compounds in the brain, as well as in the regulation of transport and detoxifying systems at the BCSFB. The knowledge hereby created has far-reaching potential for improving the challenging task of delivering therapeutic drugs into the CNS.
- Bisphenol A effects in transthyretin and barrier integrity markers in the choroid plexus: implications in amyloid beta catabolismPublication . Duarte, Ana Catarina Abreu; Lopes, Helena Tomás Marcelino; Santos, Cecília Reis Alves dosThe choroid plexus is a multifunctional tissue responsible for a wide range of homeostatic functions crucial to the central nervous system, including secretion of cerebrospinal fluid, synthesis and secretion of important peptides and regulation of the chemical substances exchange between the blood and the cerebrospinal fluid, through the blood-cerebrospinal fluid barrier. Transthyretin, a protein highly expressed and secreted by choroid plexus to the cerebrospinal fluid, is the major amyloid-beta scavenger protein, contributing to its clearance. Sex hormones, as estrogens, upregulate transthyretin expression in choroid plexus, and as a consequence its regulation may be disrupted by substances that interfere with various cellular pathways regulated by endogenous hormones, known as endocrine disruptors chemicals. The human population is exposed to many chemicals with such properties, such as bisphenol A. Therefore, the present study analysed the effects of the endocrine disruptor bisphenol A on transthyretin expression in newborn rats by Whole-Mount fluorescent staining and Western blot, and at the mRNA level by Real time RT-PCR. Moreover, the effects of beta-amyloid on transthyretin expression were also investigated using the same techniques in choroid explants of newborn and young rats. Blood-cerebrospinal fluid barrier plays an important role in the regulation of molecules movement between choroid plexus and cerebrospinal fluid, and it disruption can happen when choroid plexus functions are impaired. Thus, one purpose of this work was determine the effects of both compounds, beta-amyloid and bisphenol A, in bloodcerebrospinal fluid barrier integrity through the evaluation of some membrane protein levels present in this barrier, namely, occludin, E-cadherin, claudin-1 and zonula occludens-1. Beta-amyloid treatment in rat choroid plexus seems to trigger transthyretin upregulation, in a dose-response manner. Transthyretin mRNA levels in newborn rat choroid plexus explants increased much more than in young explants. Increased transthyretin expression levels were not correlated with secretion levels. Additionally, beta-amyloid at 1ug/mL increased reactive oxygen species production in choroid plexus. Low doses of bisphenol A affected transthyretin expression in rat choroid plexus in a non-monotonic dose response way, accordingly to data previously reported in other studies with bisphenol A. The same response profile was observed in transthyretin protein and mRNA levels measured, with higher transthyretin levels verified at 50nM of bisphenol A. As reported before with beta-amyloid treatment, also bisphenol A lead to an increase of transthyretin expression in choroid plexus cells, which was not altered with significance the secretion levels of this protein. Beta-amyloid and bisphenol A clearly influence transthyretin expression in rat choroid plexus, in a dose-response manner, and in a non-monotonic dose response, respectively. In accordance to previous reports, increasing beta-amyloid levels induced transthyretin upregulation. Increased transthyretin production by choroid plexus seems to be a protective mechanisms to avoid beta-amyloid fibrillization and consequent toxicity. Bisphenol A interfered with transthyretin expression, in both positive and negative ways. Therefore, bisphenol A levels might lead to up or down of transthyretin regulation, and consequently, leading to impairment of beta-amyloid levels in brain. Blood-cerebrospinal fluid barrier integrity might be compromised by beta-amyloid and bisphenol A injuries, which explains alteration in secretion rates of controls for treated choroid plexus explants. However, further investigation is required to analyse evolution of transthyretin expression by choroid plexus throughout life, and would be also important evaluate bisphenol A effects in blood-cerebrospinal fluid barrier protein levels, to better understand bisphenol A consequences in beta-amyloid clearance.