Pharmacoresistance in epilepsy is a major challenge to successful clinical therapy. Glucocorticoid receptor (GR) dysregulation can affect the underlying disease pathogenesis. We recently reported that local drug biotransformation at the blood-brain barrier is upregulated by GR, which controls drug-metabolizing enzymes (e.g., cytochrome P450s, CYPs) and efflux drug transporters (MDR1) in human epileptic brain endothelial cells (EPI-ECs). Here, we establish that this mechanism is influenced upstream by GR and its association with heat shock proteins/co-chaperones (Hsps) during maturation, which differentially affect human epileptic (EPI) tissue and brain endothelial cells. Overexpressed GR, Hsp90, Hsp70, and Hsp40 were found in EPI vs. NON-EPI brain regions. Elevated neurovascular GR expression and co-localization with Hsps was evident in the EPI regions with cortical dysplasia, predominantly in the brain micro-capillaries and neurons. A corresponding increase in ATPase activity (*p < 0.05) was found in the EPI regions. The GR-Hsp90/Hsp70 binding patterns indicated a faster chaperone-promoted maturation of GR, leading to its overactivation in both the tissue and EPI-ECs derived from EPI/focal regions and GR silencing in EPI-ECs slowed such GR-Hsp interactions. Significantly accelerated GR nuclear translocation was determined in EPI-ECs following treatment with GR modulators/ligands dexamethasone, rifampicin, or phenytoin. Our findings reveal that overexpressed GR co-localizes with Hsps in the neurovasculature of EPI brain, increased GR maturation by Hsps accelerates EPI GR machinery, and furthermore this change in EPI and NON-EPI GR-Hsp interaction alters with the age of seizure onset in epileptic patients, together affecting the pathophysiology and drug regulation in the epileptic brain endothelium.
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This work is supported in part by the National Institute of Neurological Disorders and Stroke/National Institutes of Health grants R01NS095825 and R01NS078307 (to CG) and the National Heart, Lung, and Blood Institute/National Institutes of Health grant R56HL139564 (to AG). We are thankful for the support of the Cleveland Clinic Center of Research Excellence in Epilepsy and Comorbidities grant award and Lerner Research Institute Recognition award 2019.
Conflict of Interest
I.N. serves on the speakers bureau and as a member of ad hoc advisory board for Eisai, Inc. None of the other authors have any potential conflict of interest to disclose. We confirm that we have read the journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
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Electronic supplementary material
List of antibodies used for immunohistochemistry; immunocytochemistry and western blot (PDF 193 kb)
General experimental outline and approaches used to study Hsp-facilitated GR regulatory process in epileptic brain tissue and endothelial cells. This includes evaluation of GR-Hsp expression, GR maturation, drug regulation, and downstream CYP-MDR expression and function in epileptic (EPI) vs non-epileptic (NON-EPI) brain tissue and respective endothelial cells isolated from both brain regions. GR silencing in EPI-ECs and subsequent effect on GR-Hsp interactions has also been studied. See main text for details. (JPG 136 kb)
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Hossain, M., Williams, S., Ferguson, L. et al. Heat Shock Proteins Accelerate the Maturation of Brain Endothelial Cell Glucocorticoid Receptor in Focal Human Drug-Resistant Epilepsy. Mol Neurobiol 57, 4511–4529 (2020). https://doi.org/10.1007/s12035-020-02043-9
- Glucocorticoid receptor
- Heat shock proteins
- Drug resistance
- Blood-brain barrier
- Nuclear translocation