Timing differences of signaling response in neuron cultures activated by glutamate analogue or free radicals

Oxidative stress and excitotoxicity are both involved in the pathogenesis of neuronal degenerative diseases like ALS. In order to compare their action, some key proteins involved in their respective signaling pathways, particularly ERK and p53, were analyzed in primary cultures of cortical neurons subjected to NMDA or H2O2 treatment. Early ERK activation was detected after NMDA treatment and was maintained during 24 h, but not after H2O2 treatment. Early p53 expression was also found after NMDA treatment but diminished later. On the other hand, it progressively increased from 6 h to 24 h after H2O2 treatment. Blocking ERK1/2 activation with the upstream inhibitor U0126 inhibited NMDA-mediated p53 expression, suggesting that ERK1/2 signals drive the cells to apoptosis under these conditions. In order to identify the initial membrane target of these neurotoxins, PAK1 was analyzed. Early increase of PAK1 expression was measured after NMDA treatment and was still present after 24 h. Conversely increased PAK1 expression was only detected 24 h after H2O2 treatment. In order to define the components through which NMDA or H2O2 induce the final elements of these pathways, p21 and c-jun, we have performed a detailed functional analysis of c-jun and p21 promoters following plasmid transfection. Both p21 and c-jun were activated after NMDA treatment, but this activation was abolished after H2O2 treatment. We conclude that NMDA induces an early effect that involves activation of p53, ERK, PAK1, p21 and c-jun. On the other hand, H2O2 induces long-term p53 expression, late expression of PAK1 without activation of p21 promoter. The timing differences of the action of these neurotoxins may explain why the presence of both compounds is needed to induce neuronal death.