title: A novel blood-brain barrier co-culture system for drug targeting of Alzheimer's disease: establishment by using acitretin as a model drug.
abstract: In the pathogenesis of Alzheimer's disease (AD) the homeostasis of amyloid precursor protein (APP) processing in the brain is impaired. The expression of the competing proteases ADAM10 (a disintegrin and metalloproteinase 10) and BACE-1 (beta site APP cleaving enzyme 1) is shifted in favor of the A-beta generating enzyme BACE-1. Acitretin--a synthetic retinoid-e.g., has been shown to increase ADAM10 gene expression, resulting in a decreased level of A-beta peptides within the brain of AD model mice and thus is of possible value for AD therapy. A striking challenge in evaluating novel therapeutically applicable drugs is the analysis of their potential to overcome the blood-brain barrier (BBB) for central nervous system targeting. In this study, we established a novel cell-based bio-assay model to test ADAM10-inducing drugs for their ability to cross the BBB. We therefore used primary porcine brain endothelial cells (PBECs) and human neuroblastoma cells (SH-SY5Y) transfected with an ADAM10-promoter luciferase reporter vector in an indirect co-culture system. Acitretin served as a model substance that crosses the BBB and induces ADAM10 expression. We ensured that ADAM10-dependent constitutive APP metabolism in the neuronal cells was unaffected under co-cultivation conditions. Barrier properties established by PBECs were augmented by co-cultivation with SH-SY5Y cells and they remained stable during the treatment with acitretin as demonstrated by electrical resistance measurement and permeability-coefficient determination. As a consequence of transcellular acitretin transport measured by HPLC, the activity of the ADAM10-promoter reporter gene was significantly increased in co-cultured neuronal cells as compared to vehicle-treated controls. In the present study, we provide a new bio-assay system relevant for the study of drug targeting of AD. This bio-assay can easily be adapted to analyze other Alzheimer- or CNS disease-relevant targets in neuronal cells, as their therapeutical potential also depends on the ability to penetrate the BBB.
authors.affiliation: REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz and European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Freese
authors.affiliation: Department of Psychiatry and Psychotherapy, Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Reinhardt
authors.affiliation: Department of Psychiatry and Psychotherapy, Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Hefner
authors.affiliation: REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz and European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Unger
authors.affiliation: REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz and European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
authors.forename: C James
authors.initials: CJ
authors.lastname: Kirkpatrick
authors.affiliation: Department of Psychiatry and Psychotherapy, Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Endres
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism; D000072197:ADAM10 Protein; D017255:Acitretin / Q000378:metabolism / Q000494:pharmacology*; D000544:Alzheimer Disease / Q000188:drug therapy; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D016503:Drug Delivery Systems; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007641:Keratolytic Agents / Q000378:metabolism / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine
chemical_list: C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D007641:Keratolytic Agents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D017255:Acitretin