RESUMO
ABC (ATP Binding Cassette) efflux transporters at the blood-brain barrier, P-glycoprotein (ABCB1), multidrug resistance associated protein 4 (ABCC4) and breast cancer resistance protein (ABCG2), are important for protecting the brain from circulating xenobiotics. Their expression is regulated by signals from surrounding brain tissue that may alter in CNS pathologies. Differences have been reported in transporter expression on brain vasculature of Alzheimer's subjects where raised levels of ß-amyloid (Aß) occur. The present study examines in vitro the effects of Aß using immortalised brain endothelial cells (hCMEC/D3). Significantly lower expression of ABCB1 but not ABCC4 or ABCG2 was found following exposure to Aß(1-42) peptide but not its scrambled equivalent. This was evident at both protein and transcript level and was reflected in lower transcriptional activity of the ABCB1 promoter as judged from the luciferase reporter gene assay and in decreases in ABCB1-mediated efflux of rhodamine 123. Aß exposure also affected Wnt/ß-catenin signalling, decreasing levels of ß-catenin protein, reducing activation of TOPFLASH and increasing transcript levels of endogenous inhibitor, Dkk-1. Application of Wnt3a reversed the Aß-induced changes to ABCB1 protein. These results suggest that Aß may impair Wnt/ß-catenin signalling at the blood-brain barrier but that activation of this pathway may restore ABCB1.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Peptídeos beta-Amiloides/farmacologia , Encéfalo/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Transformada , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Rodamina 123/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sais de Tetrazólio , Tiazóis , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Multidrug efflux transporters of the ATP-Binding cassette (ABC) family, P-glycoprotein (Pgp), multidrug-resistance associated protein 4 (MRP4) and breast cancer resistance protein (BCRP), located on endothelial cells lining brain vasculature play important roles in limiting movement of substances into and enhancing their efflux from the brain. Signals from the surrounding brain normally maintain such barrier function but these may become altered in CNS pathologies such as Alzheimer's disease (AD). Previous studies have reported decreases in the glucose transporter, Glut-1, in brain vasculature of AD patients. The present study investigates the status of the multidrug efflux transporters. Sections of frozen brain from hippocampal region obtained from male AD and age-matched non-demented cases were examined for amyloid plaques and Dkk-1 expression and subjected to dual fluorescence immunochemical staining using antibodies against Pgp, BCRP or MRP4 and von Willebrand factor. Protein expression of each transporter was assessed using confocal microscopy, quantifying peak fluorescence values of cross sectional profiles across brain microvessels. Results in brain microvessels revealed expression of Pgp protein to be significantly lower in hippocampal vessels of patients with AD compared to normal individuals whereas that of MRP4 or BCRP protein was not. By contrast, analysis of the sections at protein level via Western blotting or at transcript level by qRT-PCR did not reveal significantly lower expression for either Pgp or BCRP. Such analysis did however reveal higher than normal expression in the AD brains of MRP4, probably due to gliosis, MRP4 being present also in glial cells.
