Abstracts

Rationale: Glucocorticoid receptor (GR) association with heat shock proteins (Hsps) at blood-brain barrier (BBB) endothelium in pharmacoresistant epilepsy remains unknown. We recently reported drug biotransformation at the BBB is upregulated by GR controlling drug metabolizing enzyme, cytochrome P450s (CYPs) and efflux transporters (MDR1) in human epileptic brain endothelial cells (EPI-ECs). In this study, we determine the molecular mechanisms of GR regulation at the BBB endothelium of intractable epilepsy by exploring 1) GR and molecular chaperones/Hsps neurovascular localization patterns; 2) GR interaction pattern with Hsp90/Hsp70 in epileptogenic (EPI) vs nonepileptogenic (NONEPI) regions of the epileptic brain and EPI-ECs; and 3) endothelial GR nuclear translocation pattern following drug exposure. Methods: Surgical brain resections from focal (EPI) areas were obtained from patients (n = 21 subjects, mostly focal cortical dysplasia) with medically intractable epilepsy and compared with relatively nonfocal (NONEPI) regions of the same tissue pre-characterized by electrophysiology and histopathology. GR and Hsps were analyzed by immunoblotting and immunohistochemistry. Immunoprecipitation of GR in EPI-ECs / epileptic brain tissue was performed; GR interaction with Hsp90/ Hsp70 and ATPase activity of the chaperones were evaluated. GR nuclear translocation in EPI-ECs was followed post-exposure to dexamethasone (DEX), rifampicin (RIF) and phenytoin (PHT).  Results: Overexpression of GR (*p<0.01), Hsp90, Hsp70 and Hsp40 (*p<0.03) were found in EPI vs. NONEPI brain regions. Elevated GR neurovascular expression and co-localization with Hsps was evident in the EPI regions with cortical dysplasia and reactive gliosis, predominantly in the brain micro-capillaries and the neurons. A corresponding increase in ATPase activity (*p < 0.05) was found in the EPI regions. The GR-Hsp90/ GR-Hsp70 binding patterns indicate faster chaperone promoted maturation of GR leading to its over-activation in EPI /focal region both in the brain tissue and EPI-ECs, and accelerated nuclear translocation following treatment with DEX, RIF or PHT. Conclusions: In summary (1) overexpressed neurovascular GR co-localizes with Hsps in EPI brain; (2) increased GR-Hsp interaction and ATPase activity accelerates EPI GR machinery; (3) disease-state and drug exposure impacts brain endothelial GR regulation. Together, GR-Hsp regulatory mechanism observed could control enhanced CYP-MDR activity, limiting drug access to epileptic brain and contribute to pharmacoresistance.  Funding: This study supported in part by NIH-NINDS grant R01NS095825 awarded to Dr. Chaitali Ghosh.