Is P‐glycoprotein functionally active at the Blood‐CSF Barrier?

Abstract

The brain is protected from circulating metabolites and xenobiotics by two major barriers: the blood‐brain barrier (BBB) and the blood‐cerebrospinal fluid (CSF) barrier (BCSFB). The drug efflux transporter P‐glycoprotein (P‐gp) is known to be expressed luminally at the BBB where it limits the entry of circulating drugs and toxins into the brain. The BCSFB, formed by the choroid plexus epithelial cells, similarly expresses membrane transporters to mediate solute efflux or influx into the CSF. There are conflicting findings on P‐gp activity at the BCSFB, and limited studies of P‐gp localization posit the transporter is expressed apically or subapically, implying a paradoxical function to mediate a blood‐to‐CSF transport of xenobiotics and endobiotics. The objective of this study is to clarify the localization and function of P‐gp at the BCSFB utilizing a quantitative fluorescence microscopy approach. Calcein‐AM, commonly used to evaluate P‐gp‐mediated efflux, is a permeable, non‐fluorescent P‐gp substrate that is converted by intracellular esterases into calcein, a fluorescent and impermeable compound. Time‐dependent uptake of 250 nM calcein‐AM was performed in lateral ventricle choroid plexus isolated from adult FVB mice, and confocal imaging was utilized to observe the accumulation of calcein formed in the choroid plexus epithelial cells. Image segmentation was performed using ImageJ to individually quantify choroid plexus cell and blood capillary accumulation of calcein. Coincubation with 2 µM elacridar, a potent P‐gp inhibitor, increased accumulation of calcein in the choroid plexus cells by 5.7‐fold and increased blood capillary accumulation by 11.2‐fold, indicating inhibition of apical efflux transport. Our data suggests that that P‐gp is indeed functionally active at the apical, CSF‐facing membrane of the BCSFB. As CSF drug concentrations are frequently used as a surrogate measure of CNS drug exposure, elucidating the functional role of P‐gp at the BCSFB is critical for understanding the interplay of BBB and BCSFB transporters and their impact on CNS pharmacokinetics and pharmacodynamics.