Evidence of tumor necrosis factor receptor 1 signaling in human temporal lobe epilepsy

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.

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 pathway), 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 homeostasis and induce an intrinsic apoptosis pathway (Xu et al., 2005). There is emerging evidence that seizures trigger mitochondrial and ER dysfunction and activate Bcl-2 proteins and caspase-9, with protective effects achieved by blocking caspase-9 (Henshall et al., 2000a, Henshall et al., 2001a, Henshall et al., 2002, Kitao et al., 2001, Narkilahti et al., 2003, Yamamoto et al., 2006).

The extrinsic apoptosis pathway is initiated by oligomerization of plasma-membrane-localized death receptors of the tumor necrosis factor (TNF) superfamily (Ashkenazi and Dixit, 1998). TNF receptor 1 (TNFR1)-mediated cell death involves recruitment of TNFR-associated death domain protein (TRADD) which then binds Fas-associated death domain protein (FADD) and activates caspase-8/10 (Chinnaiyan et al., 1996, Schutze et al., 1999, Micheau and Tschopp, 2003). Other mechanisms of TNFR1-mediated cell death have been proposed including activation of the apoptosis signal-regulating kinase 1 (ASK1) and sphingomyelinase-mediated ceramide production (Wallach et al., 1999). TNFR1 can also mediate inflammatory responses, proliferation and repair which require TNFR-associated factors (TRAFs) and nuclear factor κB (NFκB) (Karin and Lin, 2002). Cross-talk between extrinsic and intrinsic pathways occurs under certain circumstances. For example, TNFα/TNFR1 can trigger intrinsic pathways via caspase-8-mediated cleavage of the Bcl-2 family protein Bid, thus prompting mitochondrial dysfunction (Gross et al., 1999) or induction of ER stress (Xu et al., 2005).

Seizure activity triggers release of TNFα (de Bock et al., 1996), and activation of TNFR1 by TNFα induces neuronal death under certain conditions (Yang et al., 2002). Seizures that damage the hippocampus induce formation of a TNFR1 signaling complex that includes TRADD and FADD, which may recruit and activate caspase-8 (Henshall et al., 2003, Shinoda et al., 2003). Surprisingly, caspase-8 activation may precede mitochondrial dysfunction after seizures in rats (Henshall et al., 2001a, Henshall et al., 2001b). Furthermore, inhibition of caspase-8 is neuroprotective against seizure damage in vivo (Henshall et al., 2001b) and in vitro (Meller et al., 2006) and may also mitigate mitochondrial dysfunction (Henshall et al., 2001b). Translational insights from studies using hippocampal tissue from patients with intractable TLE also support signaling involving TNFR1-associated proteins (Henshall et al., 2004). Since this extrinsic pathway may be an apical cascade activated by seizures, we investigated TNFR1 signaling in hippocampus from patients with intractable TLE.