The extracellular signal-regulated kinases (ERK) signalling cascade is a key pathway that mediates the NMDA receptor (NMDAR)-dependent neuronal plasticity and survival. However, it is not clear yet how NMDARs regulate ERK activity. Stimulation of the NMDARs induces a complex modification of ERK that includes both ERK activation and inactivation and depends on particular experimental conditions. Here we show that there exists a differential restriction in the regulation of ERK activity that depends on the pool of NMDAR that was activated. The synaptic pool of NMDARs activates ERK whereas the extrasynaptic pool does not; on the contrary, it triggers a signalling pathway that results in the inactivation of ERK. As a result, simultaneous activation of both extrasynaptic and synaptic NMDAR using bath application of NMDA or glutamate (a typical protocol explored in the majority of studies) produced ERK activation that depended on the concentration of agonists and was always significantly weaker than those mediated by synaptic NMDARs. Since the activation of the extrasynaptic NMDA is attributed mainly to global release of glutamate occurring at pathological conditions including hypoxic/ischaemic insults, traumas and epileptic brain damage, the reported differential regulation of ERK cascade by NMDARs provides a unique mechanism for an early identification of the physiological and/or pathophysiological consequences of NMDAR activation. The negative regulation of the ERK activity might be one of the first signalling events determining brain injury and constitutes a putative target of new pharmacological applications. The NMDA subtype of glutamate receptors (NMDAR) is a key receptor involved in the regulation of multiple processes related to synaptic plasticity including learning, memory, neuron development, spine formation, long-term potentiation (LTP) and long-term depression (LTD). The mechanisms underlying such diversity of neuronal responses to the activation of a single receptor are not known. At least some of these processes are associated with the NMDA-dependent activation of the extracellular signal-regulated kinases ERK1 and ERK2 (ERK) signalling cascade whose inhibition modifies spine formation, long-term memory, LTP and cell survival (Adams & Sweatt, 2002;Hardingham & Bading, 2003;Goldin & Segal, 2003;Thomas & Huganir, 2004). Stimulation of NMDA receptors by specific agonists (Bading & Greenberg, 1991;Kurino et al. 1995) or via an increase of synaptic A. Ivanov and C. Pellegrino contributed equally to this work. activity (Hardingham et al. 2001) results in strong ERK phosphorylation. On the other hand, applications of the high concentrations of NMDA (70-100 μm) to neuronal cultures provokes a complex effect; it induces both activation and inactivation of ERK (Chandler et al. 2001;Kim et al. 2005). Although different suggestions were put forward to explain the complex effects of NMDAR agonists on ERK activity (Chandler et al. 2001;Kim et al. 2005), none of them provided compelling evidence supporting their hypo...

Non-technical summary 'To be, or not to be' -thousands of neurons are facing this Shakespearean question in the brains of patients suffering from epilepsy or the consequences of a brain traumatism or stroke. The destiny of neurons in damaged brain depends on tiny equilibrium between pro-survival and pro-death signalling. Numerous studies have shown that the activity of the neuronal potassium chloride co-transporter KCC2 strongly decreases during a pathology. However, it remained unclear whether the change of the KCC2 function protects neurons or contributes to neuronal death. Here, using cultures of hippocampal neurons, we show that experimental silencing of endogenous KCC2 using an RNA interference approach or a dominant negative mutant reduces neuronal resistance to toxic insults. In contrast, the artificial gain of KCC2 function in the same neurons protects them from death. This finding highlights KCC2 as a molecule that plays a critical role in the destiny of neurons under toxic conditions and opens new avenues for the development of neuroprotective therapy.Abstract KCC2 is a neuron-specific potassium-chloride co-transporter controlling intracellular chloride homeostasis in mature and developing neurons. It is implicated in the regulation of neuronal migration, dendrites outgrowth and formation of the excitatory and inhibitory synaptic connections. The function of KCC2 is suppressed under several pathological conditions including neuronal trauma, different types of epilepsies, axotomy of motoneurons, neuronal inflammations and ischaemic insults. However, it remains unclear how down-regulation of the KCC2 contributes to neuronal survival during and after toxic stress. Here we show that in primary hippocampal neuronal cultures the suppression of the KCC2 function using two different shRNAs, dominant-negative KCC2 mutant C568A or DIOA inhibitor, increased the intracellular chloride concentration [Cl − ] i and enhanced the toxicity induced by lipofectamine-dependent oxidative stress or activation of the NMDA receptors. The rescuing of the KCC2 activity using over-expression of the active form of the KCC2, but not its non-active mutant Y1087D, effectively restored [Cl − ] i and enhanced neuronal resistance to excitotoxicity. The reparative effects of KCC2 were mimicked by over-expression of the KCC3, a homologue transporter. These data suggest an important role of KCC2-dependent potassium/chloride homeostasis under neurototoxic conditions and reveal a novel role of endogenous KCC2 as a neuroprotective molecule. Abbreviations DIV, days in vitro; shRNA, short hairpin RNA; RT, room temperature; TBSTD, tris-buffered saline, 0.1% Tween, 5% DMSO.

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