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.
Highlights
Alzheimer’s disease (AD) is a progressive degenerative disorder of the brain
Characterization of porcine brain endothelial cells (PBECs) in the blood-brain barrier (BBB) Co-culture Model The development of the co-culture model system of barrierbuilding brain endothelial cells with neuronal cells serving as a biological reporter system is schematically shown in Figure 1 A
In co-culture with the human neuroblastoma cell line SH-SY5Y the morphology of the PBECs was unaltered and no negative impact regarding the expression of the tight junction proteins was observed (Figure 1 B (d–f))
Summary
Alzheimer’s disease (AD) is a progressive degenerative disorder of the brain. While maximally 5% of all cases of this type of dementia are based on gene mutations [1], the cause of the sporadically occurring cases is still enigmatic. A recent study of Vos and colleagues demonstrated that subjects with preclinical AD had a higher risk for development of AD [18] Therapeutic intervention at such preclinical stages has to face the challenge of an unimpaired BBB, which basically is a tight barrier and transport of nutrients as well as drugs is highly regulated or even impaired due to cell-cell junctions and efflux transporters such as P-gp [19,20,21]. Isolated from rat, bos taurus or pig, and co-cultured with astrocytes, pericytes or even both cell types, brain microvascular endothelial cells were shown to form a tight barrier, generally demonstrated by high transendothelial electrical resistance (TEER) and low permeability coefficients [26,27,28,29]. The advantages of in vitro models are the possibility for high throughput screening, their reproducibility and more importantly, the reduction of animal experiments
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