Abstracts

Rationale: Death-associated protein kinase (DAPK), a calcium calmodulin-regulated serine/threonine protein kinase, is a key player in multiple cell-death signaling pathways. Prolonged seizures induce neuronal death by involving death receptors and calcium; thus, we studied the expression pattern of DAPK in resected temporal lobe brain tissues from pharmacoresistant epilepsy, focusing on the neurovascular unit. Methods: We used brain tissues from patients who underwent surgical resection for intractable epilepsy (n = 14), brain tumor (n= 6) and from autopsy controls (n = 4). We also used isolated primary human endothelial cells (EPI-ECs; n = 5) and glial cells (n = 5) from epileptic brain resections and compared with commercially procured control human brain microvascular endothelial cells (HBMECs, n = 5)/astrocytes (HA, n = 5). The expression of DAPK in temporal lobe tissues was evaluated by immunohistochemistry; the levels of DAPK and phosphorylated-DAPK were determined by western blot. The DAPK staining was further compared between epileptic tissue and brain tumor pathology. The subcellular localization of DAPK in epileptic brain fractions was explored. DAPK expression (mRNA and protein) in endothelial cells and astrocytes was evaluated by RT-PCR and western blot analysis. Results: DAPK over-expression was predominant across neurons, brain capillaries and astrocytes in the human epileptic brain compared to control. In contrast to autopsy brain samples, DAPK expression and overall staining pattern in epileptic brain was remarkably similar to that observed in brain tumor pathology with predominance of DAPK expression across neurons. Both DAPK and phospho-DAPK was significantly increased (p < 0.001) in microsomal fractions of human epileptic brain compared to cytoplasmic or mitochondrial fractions (7/7subjects). Although DAPK mRNA expression were same, however, a significantly increased level of DAPK and phospho-DAPK protein expression was found in EPI-ECs compared to controls. A nonsignificant difference in DAPK level in epilepsy and control astrocytes was noted. Conclusions: Together, these findings suggest that DAPK could be a potential molecular target in neuronal death in epilepsy. In addition, the epileptic endothelial cell DAPK expression identified for the first-time may have clinical relevance for future therapeutic interventions. Funding: This work is supported by NIH grants R01NS095825 and R01NS078307; Brain & Behavior Foundation (NARSAD); American Heart Association National Center Scientist Development Grant (13SDG13950015); and the Alternatives Research & Development Foundation.