Effect of lithium chloride on expression of P-glycoprotein and low density lipoprotein receptor-related protein 1 on blood-brain barrier

Background: This study investigates the role of lithium chloride (LiCl) in regulating the expression of P-glycoprotein (P-gp) and low density lipoprotein receptor-related protein 1 (LRP1) in endothelial cells derived from brain vasculature. There is evidence of a decreased expression of P-gp and LRP1 in Alzheimer’s disease (AD) patients. In addition to the reduced expression of these efflux transporters at the blood-brain barrier (BBB), there is an increased accumulation of amyloid-β (Aβ) peptides in the AD brain, and Aβ peptides are known to cause neurodegeneration. Both P-gp and LRP1 are involved in the clearance of Aβ through the BBB, and therefore, it is hypothesized that up-regulating the expression of these transporters at the BBB should lead to increased clearance and reduced Aβ accumulation in the brain. LiCl activates Wnt/β-catenin signaling by inhibiting glycogen synthase kinase 3β (GSK-3β, an inhibitory enzyme of the Wnt signaling pathway), and has been shown to protect neurons from degeneration by stabilizing the endogenous levels of β-catenin (a key transducer of Wnt signaling). Activation of Wnt/β-catenin signaling can lead to increased P-gp expression, and hence may increase the efflux of Aβ from the brain and reduce Aβ-induced neurodegeneration. The involvement of Wnt/β-catenin signaling in the regulation of P-gp expression in in-vitro models of the BBB as well as its role in regulating other properties of brain endothelial cells suggests the Wnt/β-catenin pathway may represent a promising target when developing new treatments for AD. In order to test this hypothesis, the effect of LiCl on the expression of P-gp at the mRNA and protein level and the expression of LRP1 at the mRNA level was examined in immortalized human brain endothelial (hCMEC/D3) cells. In a previously published study, LiCl was found to enhance the protein expression of P-gp, however, this was only examined at therapeutically irrelevant concentrations, and in addition, the effect of LiCl on the gene expression of P-gp was not assessed. Furthermore, the effect of LiCl on the expression of LRP1 remains to be addressed, and therefore this study was designed to examine the ability of a Wnt modulator such as LiCl to alter the expression of P-gp and LRP1 in an in-vitro BBB model. Methods: hCMEC/D3 cells (seeding density; 50000 cells/cm2) were treated with three different concentrations (1.25, 2.5 and 5.0 mM) of LiCl for 48 hours. Total RNA isolated from the LiCl and vehicle-treated cells was quantified using a Nanodrop spectrophotometer and the protein isolated from these cells was quantified through a bicinchoninic acid (BCA) protein assay. Gene expression of P-gp and LRP1 was assessed via quantitative polymerase chain reaction (qPCR) and the protein expression of P-gp was quantified through Western blotting. Both qPCR and Western blot techniques were validated to assess the fold change differences in expression of P-gp (at mRNA and protein levels) and LRP1 (mRNA level only) after LiCl treatment. Prior to experimentation, the qPCR protocol was optimized by assessing the efficiency of the primers for P-gp, LRP1 and the housekeeping gene, GAPDH. The Western blot technique was optimized by determining the loading mass of protein per well, and the desired concentrations of primary and secondary antibodies required to accurately quantify the protein expression of P-gp. Results: The efficiency of the qPCR primers for P-gp, LRP1 and GAPDH (housekeeping gene) were 102.6, 91.4 and 93.4%, respectively, which lies within the standard range (90-110%). After optimization of the Western blot technique to measure P-gp protein levels in hCMEC/D3 cells, a loading mass of 30 µg protein/well and dilutions of 1:500 and 1:15000 for the primary and secondary antibodies, respectively, were considered appropriate. The effect of LiCl on hCMEC/D3 cells was assessed at three different concentrations, and therapeutically relevant concentrations (1.25 and 2.5 mM) reduced the gene expression of P-gp by 0.27 and 0.21 fold and LRP1 by 0.33 and 0.31 fold, respectively, whereas the therapeutically irrelevant concentration of LiCl (5.0 mM) did not affect the gene expression of either transporter. When assessing the protein expression of P-gp, no significant changes were observed after LiCl treatment at 1.25, 2.5 and 5.0 mM. The expression of LRP1 at the protein level was not investigated as the low expression of LRP1 in hCMEC/D3 cells demanded an excessive loading mass (≥ 150 µg), resulting in the appearance of ‘ghost bands’. Conclusions: Based on the results of the current study, it can be concluded that LiCl reduces the mRNA expression of P-gp and LRP1 at therapeutically-relevant concentrations, but does not affect the protein expression of P-gp. Furthermore, a therapeutically irrelevant concentration of LiCl that has been used in other studies (5 mM), is ineffective in altering the gene expression of P-gp and LRP1. The inconsistent transcriptional and translational effects of LiCl on P-gp expression might be due to the effect of LiCl on other pathways involved in the regulation of P-gp, or, more simply, it may be that alterations in P-gp mRNA do not correlate with changes in the level of P-gp protein, as has been reported previously for other protein targets. Based on the literature, LiCl is not a specific modulator of the Wnt pathway and may affect other pathways in addition to Wnt/β-catenin signalling. Thus, in order to more clearly determine the role of Wnt/β-catenin signalling in modulating the expression of efflux transporters such as P-gp and LRP1, more specific modulators of the Wnt/β-catenin signalling cascade should be investigated, as these may be putative candidates for novel therapeutic interventions in AD patients.