Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures David C. Henshall,* Tomohiro Araki,* ,  Clara K. Schindler,* Sachiko Shinoda,* ,  Jing-Quan Lan* and Roger P. Simon* *Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon, USA; and  Department of Neurosurgery, Mie University School of Medicine, Tsu, Mie, Japan Abstract Death-associated protein (DAP) kinase is calcium-regulated and known to function downstream of death receptors, prompting us to examine its role in the mechanism of seizure- induced neuronal death. Brief seizures were focally evoked in rats, eliciting neuronal death within the CA3 subfield of the hippocampus, and to a lesser extent, cortex. Western blotting confirmed expression of DAP kinase within hippocampus and cortex at the predicted weight of 160 kDa. Immunohisto- chemistry revealed seizures triggered a significant increase in numbers of DAP kinase-expressing cells within CA3 and cortex, without affecting cell counts within seizure-resistant CA2 or the dentate gyrus. Numbers of DAP kinase-expressing cells were increased in relation to specific patterns of injury- causing seizure activity, electrographically defined. Seizures caused an early increase in DAP kinase binding to actin, and association with calmodulin. Co-immunoprecipitation studies also revealed seizures triggered binding of DAP kinase to the tumor necrosis factor receptor 1 and the Fas-associated death domain protein, commensurate with caspase-8 proteolysis. In contrast, within surviving fields of the hippocampus, DAP kinase interacted with the molecular chaperone 14–3)3. These data suggest DAP kinase is involved in the molecular pathways activated during seizure-induced neuronal death. Keywords: apoptosis, Bcl-2, brain, epilepsy, necrosis, neurodegeneration, p53. J. Neurochem. (2003) 86, 1260–1270. Death-associated protein (DAP) kinase is one of a five- member group of cell death regulatory proteins isolated as positive mediators of apoptosis induced by the cytokine interferon-c, using a functional cloning strategy (Deiss et al. 1995). DAP kinase is an 160-kDa calcium/calmodulin- dependent serine/threonine kinase with eight ankyrin repeats, two P-loop motifs, a cytoskeleton binding region and a death domain (Deiss et al. 1995; Cohen et al. 1997; Kimchi 2001). While the exact mechanism of action remains elusive, DAP kinase can be activated downstream of death receptors in B-cell lymphoma (Bcl)-2-/caspase-related pathways (Cohen et al. 1999; Raveh et al. 2001), and can disrupt actin microfilaments and cytoskeleton organization (Cohen et al. 1997; Inbal et al. 2002). DAP kinase has also been implicated in apoptosis induced by c-Myc/p53 and trans- forming growth factor-b (Raveh et al. 2001; Jang et al. 2002). Disruption of intracellular calcium homeostasis has long been implicated as a trigger for neurodegeneration (Choi 1988). However, recent work supports death receptor activation and subsequent death domain-mediated recruit- ment of initiator caspases as a significant contributor to neuronal death (Felderhoff-Mueser et al. 2000; Jin et al. 2001a; Qiu et al. 2002). DAP kinase is expressed throughout most brain regions in the developing rodent Received March 28, 2003; revised manuscript received May 19, 2003; accepted May 20, 2003. Address correspondence and reprint requests to Dr David C. Henshall, Robert S. Dow Neurobiology Laboratories, Legacy Clinical Research and Technology Center, 1225 NE 2nd Avenue, Portland, OR 97232, USA. E-mail: dhenshall@DowNeurobiology.org Abbreviations used: Bcl, B-cell lymphoma; DAPI, 4¢,6-diamino-2- phenylindole; DAP kinase, death-associated protein kinase; EEG, elec- troencephalogram; FADD, Fas-associated death domain protein; FITC, fluorescein isothiocyante; NMDA, N-methyl-D-aspartate; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TNFR, tumor necrosis factor receptor; TRADD, TNFR-associated death domain protein; TUNEL, terminal deoxynucleotidyl dUTP nick end labeling. Journal of Neurochemistry , 2003, 86, 1260–1270 doi:10.1046/j.1471-4159.2003.01934.x 1260 Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 1260–1270