Involvement of TNF-α in glutamate-induced apoptosis in a differentiated neuronal cell line

Abstract

We examined the involvement of tumor necrosis factor (TNF)-α on glutamate-induced cytotoxicity in a differentiated neuronal cell line. In this study, we used nerve growth factor (NGF)-differentiated PC12h cells. Glutamate cytotoxicity was assessed using the MTS and TUNEL assays. To detect TNF-α levels in culture supernatants after glutamate exposure, we used ELISA methods. The involvement of caspase-8, which is downstream from TNF receptor 1 (TNF-R1) in glutamate-induced cytotoxicity, was determined by Western blot analysis. The MTS assay showed that the addition of glutamate resulted in dose-dependent cell death, while the TUNEL assay showed that glutamate induced apoptosis in differentiated PC12h cells in a dose-dependent manner. TNF-α levels in the supernatant of glutamate-exposed cells were significantly increased compared with those in unexposed cells. In addition, glutamate caused increases in the levels of caspase-8 protein. The increases in caspase-8 levels were ameliorated by pretreatment with soluble TNF-R1. Moreover, soluble TNF-R1 significantly ameliorated the cell death induced by glutamate. These results suggest that TNF-α released from neuronal cells may be associated with glutamate-induced neuronal cell death.

Introduction

Glutamate, the principle excitatory amino acid in the central nervous system (CNS), is considered to play an important role in neurotransmission, neuronal development, and synaptic plasticity via the activation of glutamate receptors (Bleich et al., 2003, Conn, 2003). However, excessive activation of glutamate receptors, particularly of the N-methyl-d-aspartic acid (NMDA) receptor subtype, leads to neuronal cell death (Choi and Rothman, 1990). Glutamate excitotoxicity has been implicated in a number of neurological disorders, such as cerebral ischemia (Diemer et al., 1992, Nellgard and Wieloch, 1992), alcoholism (Snell et al., 1993), autoimmune encephalomyelitis (Smith et al., 2000), Alzheimer's disease (Procter et al., 1988), and glaucoma (Dkhissi et al., 1999). The mechanism by which glutamate induces neurotoxicity remains to be elucidated, although many researchers have demonstrated several candidates such as the activation of calcium-dependent enzymes (Ankarcrona et al., 1996), nitric oxide synthase (Dawson et al., 1996), and mitochondrial production of reactive oxygen species (Urushitani et al., 2001), which initiate neuronal cell death.

Tumor necrosis factor (TNF)-α is a cytokine that elicits a wide spectrum of cellular responses and has been implicated in the pathogenesis of several CNS disorders, such as multiple sclerosis (McGeer et al., 1993), autoimmune encephalomyelitis (Murphy et al., 2002), AIDS-related dementia (Troyr et al., 1995), and stroke (Clark and Lutsep, 2001). Its increased production after ischemic (Botchkina et al., 1997) and excitotoxic brain injury suggests that TNF-α has an important role in modifying the neurodegenerative process (Martin-Villalba et al., 1999, Rothwell and Hopkins, 1995). TNF-α-mediated neurotoxicity has been thought to be linked to axonal degeneration and glial changes observed in the optic nerves of patients with AIDS (Lin et al., 1997) and glaucoma (Yuan and Neufeld, 2000, Tezel et al., 2001). Moreover, TNF-α is induced by glutamate exposure in in vivo systems and it has been thought that glial cells produce TNF-α in the CNS (Lindberg et al., 2005). Thus, increased TNF-α release may play an important role in the pathogenesis of several neuronal systems including glutamate-induced neurodegeneration with the involvement of activated glial cells. However, it is unclear whether neuronal cells potentiate TNF-α production.

The objective of the present study was to investigate whether TNF-α is released from neuronal cells after glutamate exposure and whether TNF-α and its downstream molecules are involved in glutamate-mediated neuronal cell death in vitro.