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.

Historically, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis was initially considered as the only physiological and programmed form of cell death. This type of death is recurrently associated with caspases, a family of cysteine proteases activated in apoptotic conditions. However, it is now widely recognized that programmed cell death (PCD) can also occur in the complete absence of caspase activation. The existence of non-caspase PCD pathways was corroborated by the discovery of caspase-independent executioners, such as the mitochondrial protein Apoptosis-Inducing Factor (AIF). Necrosis has often been viewed as an accidental and uncontrolled cell death process. Nevertheless, increasing evidence shows that, like apoptosis, necrosis could be a highly orchestrated type of PCD. Indeed, apoptosis and necrosis present more similarities than it has been originally thought. Here, we summarize the different classifications of PCD and the current knowledge of a necrotic PCD pathway mediated by AIF: alkylating DNA-damage mediated death. We also outline the molecular mechanisms controlling this form of PCD and discuss their potential relevance in physiological and pathological settings. These emerging data on the molecular mechanisms regulating programmed necrosis may certainly have potent therapeutic consequences in treating both apoptotic-resistant tumors and degenerating adult neurons.

Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pH i , [Ca 2 þ ] i or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices-instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes. Keywords: astrocytes; energy metabolism; glycolysis; lactate; network activity; neurons INTRODUCTION High cellular energy demands during network activation are met by upregulation of cytosolic glycolysis and mitochondrial oxidative phosphorylation. Mitochondrial metabolism provides most of the ATP but glycolysis is also enhanced and may contribute to the energy production. Journal of Cerebral Blood1-4 For elucidation of the cellular basis of neuroenergetics, measurements of metabolic signals including the oxygen utilization and NAD(P)H/FAD autofluorescence provide valuable information for connecting energy metabolism with neuronal activity. NADH (reduced form) is fluorescent when excited with UV light whereas NAD þ is not, leading to a decrease in observed fluorescence as a result of NADH oxidation. In contrast, FAD (oxidized form) is fluorescent, so the oxidation of FADH 2 to FAD causes an increase in fluorescence. The fluorescence of NADH cannot be separated from that of NADPH and their emission is measured in concert (NAD(P)H). NAD(P)H fluorescence represents a 'mixed' signal since this cofactor can be produced by both glycolysis and mitochondria, whereas FAD fluorescence is entirely mitochondrial. 5,6 Measurements of these parameters in combination with electrophysiological recordings have been used in many studies to monitor the energy status during neuronal activity in brain tissues.Typically, NAD(P)H transients induced by synaptic stimulation have a characteristic biphasic waveform: the initial short dip is followed by a long-lasting overshoot. While there exists a common