Genetic variation in SLC12A5 which encodes KCC2, the neuron-specific cation-chloride cotransporter that is essential for hyperpolarizing GABAergic signaling and formation of cortical dendritic spines, has not been reported in human disease. Screening of SLC12A5 revealed a co-segregating variant (KCC2-R952H) in an Australian family with febrile seizures. We show that KCC2-R952H reduces neuronal Cl− extrusion and has a compromised ability to induce dendritic spines in vivo and in vitro. Biochemical analyses indicate a reduced surface expression of KCC2-R952H which likely contributes to the functional deficits. Our data suggest that KCC2-R952H is a bona fide susceptibility variant for febrile seizures.

The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl Ϫ concentration ([Cl Ϫ ] i ) that forms the basis for hyperpolarizing GABA A receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg 2ϩ -free solution led to a fast reduction in KCC2-mediated Cl Ϫ transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.

Functional expression of the K-Cl cotransporter KCC2 in developing central neurons is crucial for the maturation of Cl

Aims: Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD.Results: Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APPSwe/PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9–10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains.Innovation: Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity.Conclusions: We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269–1280.

A robust increase in the functional expression of the neuronal K-Cl cotransporter KCC2 during CNS development is necessary for the emergence of hyperpolarizing ionotropic GABAergic transmission. BDNF-TrkB signaling has been implicated in the developmental up-regulation of KCC2 and, in mature animals, in fast activity-dependent down-regulation of KCC2 function following seizures and trauma. In contrast to the decrease in KCC2 expression observed in the adult hippocampus following trauma, seizures in the neonate trigger a TrkB-dependent up-regulation of neuronal Cl(-) extrusion capacity associated with enhanced surface expression of KCC2. Here, we show that this effect is transient, and impaired in the hippocampus of Bdnf(-/-) mice. Notably, however, a complete absence of BDNF does not compromise the increase in KCC2 protein or K-Cl transport functionality during neuronal development. Furthermore, we present data indicating that the functional up-regulation of KCC2 by neonatal seizures is temporally limited by calpain activity.