Abstracts

Rationale: Recent evidence supports a metabolic contribution of P450 enzymes (CYP) to the drug resistant phenotype in human epileptic brain. However, the upstream molecular regulators of brain CYP expression remain unknown. We characterized the expression of pregnane xenobiotic (PXR) and glucocorticoid nuclear receptors (GR) in drug resistant epileptic brain endothelial cells and surrounding parenchyma. Methods: PXR and GR localization in human epileptic brain was evaluated by immunohistochemistry in specimens from subjects who underwent temporal lobe resections to relieve drug resistant seizures. We used primary cultures of endothelial cells obtained from epileptic brain samples (EPI-ECs, n=8), commercially available human brain microvascular endothelial cells (HBMECs; n=8) and human hepatocytes (n=3). PXR and GR mRNA levels in brain ECs were determined by cDNA microarrays. The expression of PXR and GR proteins in whole ECs lysates and sub-cellular fractions was quantified by western blotting. PXR and GR silencing was performed in human EPI-ECs and downstream CYP targets quantified. We manipulated PXR and GR expression in human brain primary culture to measure the downstream effects on CYP expression. Results: In epileptic brain, robust PXR and GR expression was confined to the neurovascular unit. Hepatocytes were characterized by the highest levels of PXR and GR expression and were thus used as a positive control. Increased nuclear receptor mRNA levels were found in EPI-ECs as compared to control HBMECs. Increased PXR and GR protein expression was also found in EPI-ECs. We found expression of PXR and GR in both cytoplasmic and nuclear sub-cellular endothelial cell fractions; a significant increase of PXR/GR in these fractions was found in EPI-ECs compared to controls. CYP3A4, 2C9, and 2E1 were overexpressed in EPI-ECs compared to control; in contrast, CYP2D6 and CYP2C19 were down-regulated or absent in EPI-ECs. PXR silencing in EPI-ECs showed decreased expression of CYP3A4 (4/4 EPI-ECs) and CYP2E1 (3/4 EPI-ECs), while CYP2C9 levels were unchanged when compared to non-silenced EPI-ECs counterparts. However, GR silencing in EPI-ECs showed decreased expression of CYP3A4 (4/4 EPI-ECs) and CYP2C9 (4/4 EPI-ECs) expression when compared to non-silenced EPI-ECs. Conclusions: Our results indicate over-expression of PXR and GR in endothelial cells derived from human epileptic brain. A differential involvement of PXR or GR and CYPs may be responsible for altered drug metabolism instigated by abnormal CYP regulation in epileptic BBB endothelial cells. Funding: The work is supported by R01NS078307 (awarded to NM, DJ and CG). UH4TR000491, awarded to DJ and CG. R01NS43284, R41MH093302, R42MH093302, and R21HD057256 awarded to DJ. Brain and Behavior Foundation (NARSAD); American Heart Association (13SDG13950015) and Alternative Research Development Foundation Grant (ARDF), awarded to CG.