Elsevier

Neurobiology of Aging

Volume 32, Issue 8, August 2011, Pages 1477-1485
Neurobiology of Aging

Age-dependent decline of blood–brain barrier P-glycoprotein expression in the canine brain

https://doi.org/10.1016/j.neurobiolaging.2009.08.014Get rights and content

Abstract

The efflux transporter P-glycoprotein serves as a major molecular gatekeeper at the blood–brain barrier. It has been suggested that a reduction of P-glycoprotein activity with aging might enhance exposure of brain tissue to exogenous and endogenous compounds thereby contributing to the development of neurodegenerative diseases.

Brain tissue from owner-kept dogs renders an excellent tool to study the impact of aging on the background of variable environmental and genetic influencing factors. Therefore, we determined expression rates of P-glycoprotein in canine post-mortem tissue from 23 non-laboratory dogs. P-glycoprotein expression in the parahippocampal cortex exhibited a negative correlation with age. Analysis of the area labeled for P-glycoprotein in dogs aged >100 months revealed a 72% drop in P-glycoprotein expression as compared to young adults aged 23–36 months. Respective data from the dentate hilus and dentate gyrus indicated an earlier drop with a reduction by 77 and 80% in dogs aged 37–99 months in comparison with younger individuals. In contrast to the decline observed with aging in dogs without plaques, P-glycoprotein expression rates rather tended to increase with further aging in dogs with plaque formation.

In conclusion, the thorough analysis of P-glycoprotein expression rates in non-laboratory dogs revealed a significant decline with aging. The data strongly support the concept that age-dependent changes might predispose to neurodegenerative diseases. In the early pathogenesis of Alzheimer's disease which is modelled by diffuse plaques in the canine brain, an up-regulation of P-glycoprotein might act as a compensatory mechanism to enhance Abeta efflux from the brain. Future studies are necessary to further evaluate the correlation between Abeta deposits and P-glycoprotein expression in different phases of the disease.

Introduction

The blood–brain barrier (BBB) is a physical gate that controls the passage of compounds from the blood to the central nervous system (CNS). On a molecular level, efflux transporters localized in the luminal membrane of brain capillary endothelial cells significantly contribute to barrier function (Loscher and Potschka, 2005a). Several studies pointed to a predominant role of the efflux transporter P-glycoprotein as a major gatekeeper in the BBB (Fromm, 2004, Schinkel, 1999). P-glycoprotein comprises more than one substrate binding domain allowing for a broad substrate spectrum including several pharmaceutical compounds and other xenobiotics.

Expression rates and the functional state of P-glycoprotein are relevant for several reasons. First, BBB efflux transport can significantly limit brain penetration and efficacy of CNS therapeutics, thereby, contributing to therapeutic failure (Loscher and Potschka, 2005a). Second, low expression levels or lack of functional P-glycoprotein enhances the sensitivity towards xenobiotics contributing not only to acute intoxications but also to an increased long-term risk for specific diseases including neurodegenerative diseases (Lee and Bendayan, 2004).

Expression of efflux transporters is regulated in a highly dynamic manner (Miller et al., 2008). This regulatory process can be considered as a mechanism that allows an adaptation to changing requirements in detoxification and tissue protection. Regarding the physiological changes in P-glycoprotein expression, it has been recently suggested that aging might affect basal P-glycoprotein levels (Mangoni, 2007). Positron emission tomography studies using the P-glycoprotein substrate radiotracer (R)-[(11)C]verapamil gave first evidence indicating a decline in P-glycoprotein activity during aging (Bartels et al., 2008a, Toornvliet et al., 2006). To our knowledge no data are available regarding a correlation between aging and P-glycoprotein expression.

Therefore, we studied P-glycoprotein expression rates in post-mortem tissue from dogs including all stages of life from young adults up to aged individuals. Tissue from owner-kept dogs which were exposed to different environmental conditions throughout their life and which differ by their genetic background renders an excellent tool to study the impact of aging on the background of other variable influencing factors (Pekcec et al., 2008). Moreover, aged dogs can develop spontaneous amyloid-beta (Abeta) protein deposits (Czasch et al., 2006). Thus, canine post-mortem tissue offers the possibility to study a correlation between these Abeta deposits, which model early stages of Alzheimer's disease, and P-glycoprotein expression. This aspect is of specific interest as low P-glycoprotein expression levels are considered as a contributing factor in the pathophysiology, development, and progression of Alzheimer's disease (Deane and Zlokovic, 2007, Lee and Bendayan, 2004).

Section snippets

Brain tissue

Brain tissues of 24 dogs aged from 22 to 216 months were used in this study and processed as described previously (Czasch et al., 2006, Pekcec et al., 2009). In short: immediately after death brains were removed and fixed in 10% formalin for 10 days, cut in transverse sections and embedded in paraffin wax. Five micrometer thick transversal sections containing the hippocampus and adjacent cortical tissue were mounted on positively charged slides (Superfrostplus, Menzel-Gläser, Braunschweig,

Age-dependent decline of P-glycoprotein expression

Immunolocalization of the P-glycoprotein-antibody C219 (Calbiochem, Darmstadt, Germany) was observed in microvessel endothelial cells of all dogs which were euthanized or died due to disease not affecting the brain (Fig. 1A and B) (Table 1). Expression in endothelial cells was substantiated by double-labeling with the endothelial cell marker Glut-1 which unambiguously demonstrated a co-localization of Glut-1 and P-glycoprotein (Fig. 3A).

No distinct P-glycoprotein staining of parenchymal cells

Discussion

The analysis of BBB P-glycoprotein expression rates in post-mortem tissue from adult non-laboratory dogs revealed a significant decline associated with the process of aging. Earlier studies on age-related changes in BBB P-glycoprotein expression have especially focused on the course during embryogenesis and the early postnatal phase of laboratory rodents (Matsuoka et al., 1999, Tsai et al., 2002). Studies in rats demonstrated that P-glycoprotein becomes detectable from postnatal day 7 and then

Conflicts of interest

The authors disclose any actual or potential conflicts of interest

Disclosure statement

We verify that all pet owners approved the use of post-mortem tissue of their pets for pathological evaluation and scientific use.

Acknowledgments

We thank Petra Grünig for excellent technical assistance. This research was supported by a grant DFG PO 681/4-1 (to HP) from the German Research Foundation.

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