Evidence of tumor necrosis factor receptor 1 signaling in human temporal lobe epilepsy Akitaka Yamamoto a,b , Clara K. Schindler a , Brona M. Murphy c , Carmen Bellver-Estelles c , Norman K. So d , Waro Taki b , Robert Meller a , Roger P. Simon a , David C. Henshall a,c, ⁎ a Robert S. Dow Neurobiology laboratories, Legacy Research, Portland, OR, USA b Department of Neurosurgery, Mie University School of Medicine, Tsu, Mie, Japan c Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland d Oregon Comprehensive Epilepsy Program, Neurological Sciences Center, Portland, OR, USA Received 3 May 2006; revised 28 June 2006; accepted 1 July 2006 Available online 17 August 2006 Abstract Seizures, particularly when prolonged, may cause neuronal loss within vulnerable brain structures such as the hippocampus, in part by activating programmed (apoptotic) cell death pathways. Experimental modeling suggests that seizures activate tumor necrosis factor receptor 1 (TNFR1) and engage downstream pro- and anti-apoptotic signaling cascades. Whether such TNFR1-mediated signaling occurs in human temporal lobe epilepsy (TLE) is unknown. Presently, we examined this pathway in hippocampus surgically obtained from refractory TLE patients and contrasted findings to matched autopsy controls. Western blotting established that total protein levels of the TNFR1 proximal signaling adaptor TNFR-associated protein with death domain (TRADD), cleaved initiator caspase-8 and apoptosis signal-regulating kinase 1 (ASK1) were higher in TLE samples than controls. Intracellular distribution analyses revealed raised cytoplasmic levels of TNFR1, TRADD and the caspase-8 recruitment adaptor Fas-associated protein with death domain (FADD), and higher levels of TRADD and cleaved caspase-8 in the microsomal fraction, in TLE samples. Immunoprecipitation studies detected TRADD–FADD binding, and fluorescence microscopy revealed TRADD co- localization with FADD in TLE hippocampus. These data suggest that TNFR1 signaling is engaged in the hippocampus of patients with refractory temporal lobe epilepsy. © 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Brain; Endoplasmic reticulum; Epileptogenesis; Neuroinflammation; Seizure Introduction Seizures, particularly when prolonged, carry the potential of harming the brain. In addition to the recognized impact of status epilepticus on brain, neuroimaging studies suggest that refractory temporal lobe epilepsy (TLE) patients who continue to experience seizures may also be at risk of ongoing structural damage (Kalviainen et al., 1998; Briellmann et al., 2002; Fuerst et al., 2003). In turn, this injury may contribute to reported progressive cognitive impairments (Jokeit and Ebner, 1999; Stefan and Pauli, 2002). Accordingly, there is interest in delineating the mechanisms by which seizures injure brain for both acute neuroprotection or as potential avenues toward anti- epileptogenesis (Pitkanen and Sutula, 2002). Programmed (apoptotic) cell death pathways have been implicated in several neurodegenerative and neurological disorders, including TLE (Henshall and Simon, 2005). Dysfunction of mitochondria, which may be triggered by intracellular calcium overload downstream of glutamatergic neurotransmission, leads to release of cytochrome c and activation of the cysteine protease caspase-9 (intrinsic path- way), events regulated in part by the Bcl-2 family proteins (Liou et al., 2003; Danial and Korsmeyer, 2004). Endoplasmic reticulum (ER) stress can also result from loss of calcium Experimental Neurology 202 (2006) 410 – 420 www.elsevier.com/locate/yexnr ⁎ Corresponding author. Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 St. Stephen's Green Dublin 2, Ireland. Fax: +353 1 402 2447. E-mail address: dhenshall@rcsi.ie (D.C. Henshall). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.07.003