Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival

Abstract

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.

The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines

“…KCC2 suppression may increase chloride concentration (20) and thereby reduce GABAergic signaling. This may subsequently lead to scaling of excitatory synapses through homeostatic plasticity (21).…”

WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons

“…This experiment provides an indication of the rate of Cl − extrusion (39). The I GABAA were induced by brief focal applications of isoguvacine, a GABA A R agonist, at a holding potential (V h ) of −80 mV, whereas Cl − loading was obtained using the combination of a 10-s isoguvacine pulse and neuron depolarization to −20 mV.…”

“…shRNA KCC2 in mU6-pro vector was described previously (39) and targeted the following sequence in rat KCC2 mRNA: 5′-GACATTGGTAA-TGGAACAACG-3′ (NP_599190). A control construct with a scrambled sequence (GATGAACCTGATGACGTTC) lacked homology to any known mammalian mRNAs.…”

“…Before electrophysiology or imaging experiments, we specifically studied the efficacy of neuronal cotransfection with mixtures of pcDNAs in different proportions as described in (39). A mixture containing 0.15 + 0.6 + 0.6 mg for cotransfection of three constructs (marker + shRNA + KCC2 or marker + WNK1 + KCC2) ensured the expression of both constructs of interest into the eGFP-positive or Cl-Sensorpositive neurons or N2a cells.…”

Molecular probes and switches for functional analysis of receptors, ion channels and synaptic networks

“…A sensor with improved sensitivity to chloride, called Cl-Sensor (Markova et al, 2008), has previously been used to measure [Cl − ] i in different cell types and to analyse the function of the potassium-chloride transporter KCC2 in hippocampal neurons (Bregestovski et al, 2009;Waseem et al, 2010;Pellegrino et al, 2011). In this issue, improved methods for the stable, longlasting ratiometric recording of [Cl − ] i are described, and these provide a technique for monitoring Cl-Sensor fluorescence in different cell types using conventional fluorescence microscopy set-ups (Friedel et al, 2013).…”