Abstracts

Rationale: Blood-brain barrier (BBB) endothelial cell dysfunction has been observed in patients with drug-resistant epilepsy. Recently we identified a potential link between DNA methylation and glucose transporter, GLUT1 dysfunction in focal cortical dysplasia in human epilepsy. Autophagy, an intracellular catabolic process, plays a crucial role in removing protein aggregates and damaged intracellular organelles by transporting them to lysosomes, thereby maintaining cellular homeostasis. However, overactive autophagy can be detrimental to cell function, and that there may be an underlying mechanism between autophagy and BBB endothelial cell (EPI-EC) function in epilepsy which is understudied and therefore being investigated.

Methods: We compared surgically resected epileptic human brain tissues from focal/epileptic and non-focal/non-epileptic regions of the same brain (n=12), evaluated autophagy markers, ATG16L1, p62, SIRT2 and LC3b by western blot and determined their interaction to ATG16L1 (n=5) along with GLUT1 and lactate transporter (MCT2) by immunoprecipitation and western blot. Autophagic markers expression was compared within EPI-EC and control EC (HBMEC). To understand the link of autophagy to methylation and GLUT1 trafficking as “proof-of concept” validation, the effect of a DNA methylation inhibitor, 5-Aza-2′-deoxycytidine (5Aza/decitabine, 10µM) was tested on GLUT1 and lysosomal associated membrane protein 1 (LAMP1) localization pattern in EPI-ECs, post-5Aza treatment for 24h. The expression of autophagic markers and BBB tight-junction protein, Claudin 5 and cell-surface marker, Caveolin-1 were further compared within EPI-EC and control EC by immunocytochemistry.

Results: We found overexpression of autophagic markers p62, SIRT2 and LC3b in epileptic/ lesional brain compared to non-epileptic tissues. ATG16L1 targeted autophagic regulation of p62 and LC3b and interaction of ATG16L1 with GLUT1 and MCT2 in human epileptic focal brain tissues. DNA methylation inhibition with 5Aza in EPI-ECs decreased autophagic marker (ATG16L1, p-62, SIRT2 and LC3b I/II) levels, improved GLUT1 membrane localization with minimal GLUT1 and LAMP1 cellular overlap, and elevated Claudin 5 and Caveolin 1 levels.

Conclusions: In summary, the results suggest that focal human epileptic brain tissues and EPI-ECs have overactive expression of autophagic markers and interaction with ATG16L1. Inhibition of DNA methylation in EPI-EC 1) decreased p62 and LC3b autophagic marker 2) restored the GLUT1 membrane localization even under hypometabolic condition, and 3) increased tight-junctional and cell-surface protein levels which are critical for cellular physiology and BBB endothelial cell function, implying an association of DNA methylation and autophagy in focal epilepsies.


Funding: NA