Elsevier

Brain Research Bulletin

Volume 84, Issue 1, 15 January 2011, Pages 94-102
Brain Research Bulletin

Research report
Involvement of IRE1α signaling in the hippocampus in patients with mesial temporal lobe epilepsy

https://doi.org/10.1016/j.brainresbull.2010.10.004Get rights and content

Abstract

Cumulative evidence suggests that programmed cell death (apoptosis) may contribute to the progressive hippocampal sclerosis seen in patients with refractory mesial temporal lobe epilepsy (MTLE). The endoplasmic reticulum (ER) stress-mediated cell apoptotic pathway has recently emerged as a vital intrinsic pathway, but the molecular mechanisms underlying this process in the epileptic brain remain unclear. We investigated inositol-requiring protein 1α (IRE1α)-mediated ER stress pro-and anti-apoptotic signaling pathways in resected hippocampi from 32 patients with intractable MTLE. Immunoreactivity for the ER stress markers glucose-regulated proteins 78 and 94 was significantly higher in MTLE hippocampi than in controls. The levels of IRE1α, tumor necrosis factor receptor associated factor 2 (TRAF2), apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK), which together constitute the IRE1α/TRAF2/ASK1/JNK pro-apoptotic signaling pathway, were significantly upregulated in patients with MTLE. Immunoreactivity for caspase-4, a homologue of caspase-12 that is possibly activated by IRE1α via TRAF2 following ER stress, and caspase-3 which was a downstream effector of caspase-4, were both detected in MTLE tissue samples. In contrast, immunoreactivity for caspase-4 and caspase-3 were low or absent in control samples. Simultaneously, the X-box binding protein 1 (XBP1), a basic leucine zipper (bZIP) family transcription factor downstream of IRE1α which can promote cell survival by upregulation of multiple ER-targeted genes, was also overexpressed and activated in MTLE hippocampi. Our data suggest that chronic epilepsy is associated with ER stress, as well as induction of both IRE1α-mediated pro- and anti-apoptotic signaling pathways.

Introduction

Temporal lobe epilepsy (TLE) is the most common form of chronic focal epilepsy and the most frequent type of refractory epilepsy [60]. Mesial TLE (MTLE) is the most common subtype of TLE accompanied by mesial temporal sclerosis (MTS) [3]. Imaging and histological studies on the brains of patients with MTLE suggest that MTLE can potentially result in structural brain damage [3], [4], [5], which may be associated with progressive cognitive impairment [15], [40]. Experimental modeling has also demonstrated that repeated brief seizures in animals can induce hippocampal neuron loss and proportional cognitive deficits [29]. Accordingly, it is important to explore the mechanisms by which seizures injure the brain, the understanding of which will provide new strategies for the development of neuroprotective interventions to be used as adjuncts to anticonvulsant therapy after certain seizures in patients with refractory TLE [45].

Accumulating evidence has shown that apoptotic cell death (apoptosis) contributes to brain damage following seizures [16]. Following seizure activity, mitochondrial-dependent intrinsic cell death pathways may be triggered by high levels of intracellular calcium downstream of glutamatergic neurotransmission. The activation of these pathways may cause the release of cytochrome c from mitochondria and activation of caspase cascades [17]. Seizures may also activate extrinsic pathways through activation of tumor necrosis factor receptor 1 (TNFR1), caspase-2, and caspase-8 both in vivo and in vitro [21], [41], [61]. Both pathways are regulated and executed, in part, by the Bcl-2 family of pro- and anti-cell death genes, and the caspase family of cell death proteases [36], [19]. Recently, the endoplasmic reticulum (ER), a major organelle for mediating cell stress responses, has emerged as a key instigator of the intrinsic apoptotic pathway, involved in several neurodegenerative and neurological disorders [48], including TLE [20], [60]. However, the role of the ER stress-mediated cell apoptotic pathway in epilepsy remains largely unexplored.

Seizures induce ER stress, which likely results from loss of calcium homeostasis [19]. Inositol-requiring protein 1 α (IRE1α), an ER stress sensor protein, is believed to play an important role in ER stress [34], [35]. In response to ER stress, IRE1α dissociates from the immunoglobulin binding protein/glucose-regulated protein 78 (Bip/GRP78), and oligomerizes in the plane of the ER membrane, which in turn activates the kinase and endoribonuclease functions present in its cytosolic domain [13], [34], [51]. Activated IRE1α converts X-box binding protein 1 (XBP1) pre-mRNA into mature mRNA by unconventional splicing named XBP1s (spliced XBP1) [6]. XBP1s promotes the transcription of ER-associated degradation (ERAD) component genes [25], [55], as well as the expression of ER chaperones such as GRP78 and glucose-regulated protein94 (GRP94) [30], [53].

Severe or prolonged ER-stress triggers the apoptotic IRE1α signaling pathway [33]. The active phosphorylated form of IRE1α interacts with tumor necrosis factor receptor associated factor 2 (TRAF2) and subsequently recruits apoptosis signal-regulating kinase 1 (ASK1). The IRE1α-TRAF2-ASK1 complex formed during ER stress then activates c-Jun N-terminal kinase (JNK), which is known to influence the cell death machinery via the regulation of Bcl-2 family proteins [8], [57].

Another proposed pro-apoptotic signaling pathway involves the activation of caspase-12 (in murine) or caspase-4 (in human) by active IRE1α [62], [22]. It has been reported that the human caspase-12 gene contains several mutations, which render it functionally inactive [12]. Presently, human caspase-4 is thought to be a counterpart of murine caspase-12 [22], [44]. Although recent research has shown that IRE1α signaling plays a very important role in regulating ER stress, it is unclear whether IRE1α signaling is involved in brain injury associated with MTLE. In the current study, we show that chronic epileptic seizures induce ER stress and neuronal injury in the brains of MTLE patients, which occurs through the involvement of ER stress-mediated IRE1α signaling.

Section snippets

Human brain samples

Thirty-two MTLE patients with mesial temporal sclerosis (as assessed by MRI studies and neuropathology after surgery), who had undergone resection of the left or right anterior temporal lobe and hippocampus between January 2008 and June 2009 in the First Affiliated People's Hospital of Shanghai JiaoTong University and Shanghai Quyang Hospital, were included in this study. Clinical data are shown in Table 1. Informed and written consent was obtained from all patients or their relatives with

Upregulation of GRP78 and GRP94: evidence of endoplasmic reticulum stress in the hippocampi of MTLE with MTS patients

To determine whether ER stress was induced by recurrent seizures in the brains of patients with MTLE with MTS, we examined the expression of GRP78 and GRP94, protein markers of ER stress, in brain protein extracts using western blotting analysis (Fig. 1A). Optical density analysis, after normalization with GAPDH, showed increased expression of GRP78 and GRP94 in epileptic tissue compared with control tissue (Fig. 1B; Table 3; p < 0.01).

To support these findings, we performed GRP78 and GRP94

Discussion

The ER is an important subcellular organelle that is responsible for the proper folding and sorting of proteins. Multiple physiological or pathological condition such as glucose deprivation, perturbation of calcium homeostasis, and exposure to free radicals can cause ER stress [9]. In response to ER stress, several signaling pathways, collectively termed as the unfolded protein response (UPR), are initiated to cope with this adverse situation [34], [64]. One major pathway of UPR is the

Acknowledgements

We are grateful to all of the subjects who kindly agreed to participate in this study. This work was supported by a grant from National Natural Science Foundation of China (30870880).

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