Nitric oxide is the primary mediator of cytotoxicity induced by GSH depletion in neuronal cells

Abstract

Glutathione (GSH) levels progressively decline during aging and in neurodegenerative disorders. However, the contribution of such event in mediating neuronal cell death is still uncertain. In this report, we show that, in neuroblastoma cells as well as in primary mouse cortical neurons, GSH decrease, induced by buthionine sulfoximine (BSO), causes protein nitration, S-nitrosylation and DNA strand breaks. Such alterations are also associated with inhibition of cytochrome c oxidase activity and microtubule network disassembly, which are considered hallmarks of nitric oxide (NO) toxicity. In neuroblastoma cells, BSO treatment also induces cell proliferation arrest through the ERK1/2-p53 pathway that finally results in caspase-independent apoptosis, as evident from the translocation of apoptosis-inducing factor from mitochondria towards nuclei. A deeper analysis of the signaling processes indicates that the NO-cGMP pathway is involved in cell proliferation arrest and death. In fact, these events are completely reversed by L-NAME, a specific NO synthase inhibitor, indicating that NO, rather than the depletion of GSH per se, is the primary mediator of cell damage. In addition, the guanylate cyclase (GC) inhibitor LY83583 is able to completely block activation of ERK1/2 and counteract BSO toxicity. In cortical neurons, NMDA (N-methyl-D-aspartic acid) treatment results in GSH decrease and BSO-mediated NO cytotoxicity is enhanced by either epidermal growth factor (EGF) or NMDA. These findings support the idea that GSH might represent the most important buffer of NO toxicity in neuronal cells, and indicate that the disruption of cellular redox buffering controlled by GSH makes neuronal cells susceptible to endogenous physiological flux of NO.

p53 Orchestrates the PGC-1α-Mediated Antioxidant Response Upon Mild Redox and Metabolic Imbalance

“…L-NAME prevented PGC-1a induction in p53-transfected cells, confirming the central role of p53 in the signaling pathway that leads to upregulation of PGC-1a upon increased NO. We previously demonstrated that upon BSO treatment, ERK1/2 is upstream of p53 activation (1). Figure 2D shows that suppression of ERK1/2 activity by U0126 led to a significant reduction of BSO-mediated PGC-1a accumulation, demonstrating that PGC-1a is a downstream target of the ERK1/2-p53 signaling axis.…”

“…We have previously showed that in SH-SY5Y neuroblastoma cells GSH depletion obtained by l-buthionine sulfoximine (BSO) treatment results in the NO-extracellularregulated kinase 1/2 (ERK1/2)-mediated increase of p53 at early stages of treatment (starting at 3 h up to 15 h) (1). Here, we investigated whether this event was associated with changes in PGC-1a expression.…”

“…For these reasons, p53, NFE2L2, and PGC-1a may have overlapping functions as regulators of the antioxidant defense system. Intriguingly, NFE2L2, PGC-1a, and p53 can be activated by oxidative stress through the same redox-dependent pathways, including those managed by the nitric oxide (NO)/cGMP (1,8,23). Then, it could be hypothesized that a cross-talk exists between them upon redox imbalance.…”

“…We recently demonstrated that the disruption of the cellular redox buffer controlled by glutathione (GSH) increases endogenous physiological flux of NO in neuronal cells, leading to an upregulation of p53 by the NO/cGMP signaling pathway (1). GSH is the major cellular nonenzymatic antioxidant (21,48) playing important roles in nutrient metabolism, and regulation of many cellular events (48).…”

The role of nNOS and PGC-1α in skeletal muscle cells

“…In our laboratory, we have shown that, in skeletal muscle, caloric restriction also increases NO bioavailability independently of NOS upregulation (Aquilano et al, 2013a). In particular, caloric restriction reduces the level of GSH, the main intracellular NO buffer (Aquilano et al, 2011a;Aquilano et al, 2011b;Baldelli et al, 2008). The resulting increased NO bioavailability is the genuine mediator of the upregulation of PGC-1a, which, in turn, favours the expression of antioxidant proteins SOD2 and c-GCS (also known as glutamate cysteine ligase) (Aquilano et al, 2013a).…”