Necrostatin‐1 protects against glutamate‐induced glutathione depletion and caspase‐independent cell death in HT‐22 cells

Abstract

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death) and inhibits the integration of Bcl-2/ adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.

Neuroprotection requires the functions of the RNA-binding protein HuR

“…The flow cytometric detection of Annexin-V (AnxV) and propidium iodide (PI) suggested that all HT-22 transfectants followed a necrotic fate (AnxV + /PI + and AnxV − /PI + ) rather than an apoptotic fate after glutamate challenge; this necrotic response was clearly enhanced following the knockdown of HuR (Figure 4a). The pancaspase inhibitor zVAD-FMK did Figure 4f); these observations confirmed that apoptosis is not the predominant form of death response in these cells [28][29][30] nor it comes into effect because of HuR's loss. In contrast, a beneficial effect of the RIP-kinase 1 inhibitor necrostatin-1 (Nec-1) in HT-22 cells 29 was partially maintained in HuR lo cells, suggesting that necrotic/necroptotic pathways are maintained in the absence of HuR (Figure 4b).…”

“…The pancaspase inhibitor zVAD-FMK did Figure 4f); these observations confirmed that apoptosis is not the predominant form of death response in these cells [28][29][30] nor it comes into effect because of HuR's loss. In contrast, a beneficial effect of the RIP-kinase 1 inhibitor necrostatin-1 (Nec-1) in HT-22 cells 29 was partially maintained in HuR lo cells, suggesting that necrotic/necroptotic pathways are maintained in the absence of HuR (Figure 4b).…”

AIF‐mediated caspase‐independent necroptosis: A new chance for targeted therapeutics

“…Consequently, RIP1-deficient MEFs are resistant to MNNG-induced PCD (55). The relationship between RIP1 and AIF has been fully confirmed, by using necrostatin-1 inhibitor in other necroptotic systems, such as retinal detachmentinduced photoreceptor necrosis or glutamate-induced oxytosis in hippocampal HT-22 cells (71,72).…”

“…More recently, new forms of necroptosis have been unveiled (67)(68)(69)(70)(71). One of them is the pathway scrutinized in our laboratory: alkylating DNA damage (MNNG)-mediated PCD (11,33,39,51).…”

Necrostatin Decreases Oxidative Damage, Inflammation, and Injury after Neonatal HI

“…There is no known direct antioxidant effect of necrostatin, but it does modulate redox mechanisms in experimental systems (Xu et al, 2007). Necrostatin increases glutathione levels and decreases ROS production (Xu et al, 2007), blocks nitric oxide-mediated cell necrosis and mitochondrial ROS production (Davis et al, 2010) and may also delay the opening of mitochondrial permeability transition pore (Lim et al, 2007). These effects may be reflected in the decrease in protein oxidation in the first 24 hours and the subsequent delayed appearance of neuroprotection after HI and necrostatin administration in our model.…”