Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Slc4a10 gene, which encodes the Na ؉ -coupled Cl ؊ -HCO3 ؊ exchanger Slc4a10 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4a10 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4a10 also was expressed in neurons. Within the hippocampus, the Slc4a10 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridineinduced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure.cerebrospinal fluid ͉ epilepsy ͉ ion transport ͉ knockout mouse ͉ pH regulation

Astrocytes are critical participants in synapse development and function, but their role in synaptic plasticity is unclear. Eph receptors and their ephrin ligands have been suggested to regulate neuron-glia interactions and EphA4-mediated ephrin reverse signaling is required for synaptic plasticity in the hippocampus. Here we show that long-term potentiation (LTP) at the CA3-CA1 synapse is modulated by EphA4 in the postsynaptic CA1 cell and by ephrinA3, a ligand of EphA4 that is found in astrocytes. Lack of EphA4 increases the levels of glial glutamate transporters and ephrinA3 modulates transporter currents in astrocytes. Pharmacological inhibition of glial glutamate transporters rescues the LTP defects in EphA4 and ephrinA3 mutant mice. Transgenic overexpression of ephrinA3 in astrocytes reduces glutamate transporter levels and produces focal dendritic swellings possibly caused by glutamate excitotoxicity. These results suggest that EphA4/ephrinA3 signaling is a critical mechanism for astrocytes to regulate synaptic function and plasticity.

Blocking dual leucine zipper kinase slows disease progression in animal models of ALS and Alzheimer’s disease.

A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca 2ϩ transients were attenuated in mice deficient for the Na-K-2Cl cotransporter NKCC1. Correlated Ca 2ϩ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl Ϫ /HCO 3 Ϫ exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl Ϫ accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses.

Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo‐ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K‐Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABAA‐receptor Cl− channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl−]i roughly two‐fold. The reduced Cl− gradient nearly abolished GABA‐induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long‐term phase learning of the vestibulo‐ocular reflex, whereas baseline performance, short‐term gain‐decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning.

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1 -/-mice, decreased in Kcc3 -/-mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1 -/-or Kcc3 -/-mice were of normal density, rbc of Kcc1 -/-Kcc3 -/-mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1 -/-Kcc3 -/-SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K + content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

The c-Jun--terminal kinase (JNK) signaling pathway regulates nervous system development, axon regeneration, and neuronal degeneration after acute injury or in chronic neurodegenerative disease. Dual leucine zipper kinase (DLK) is required for stress-induced JNK signaling in neurons, yet the factors that initiate DLK/JNK pathway activity remain poorly defined. In the present study, we identify the Ste20 kinases MAP4K4, misshapen-like kinase 1 (MINK1 or MAP4K6) and TNIK Traf2- and Nck-interacting kinase (TNIK or MAP4K7), as upstream regulators of DLK/JNK signaling in neurons. Using a trophic factor withdrawal-based model of neurodegeneration in both male and female embryonic mouse dorsal root ganglion neurons, we show that MAP4K4, MINK1, and TNIK act redundantly to regulate DLK activation and downstream JNK-dependent phosphorylation of c-Jun in response to stress. Targeting MAP4K4, MINK1, and TNIK, but not any of these kinases individually, is sufficient to protect neurons potently from degeneration. Pharmacological inhibition of MAP4Ks blocks stabilization and phosphorylation of DLK within axons and subsequent retrograde translocation of the JNK signaling complex to the nucleus. These results position MAP4Ks as important regulators of the DLK/JNK signaling pathway. Neuronal degeneration occurs in disparate circumstances: during development to refine neuronal connections, after injury to clear damaged neurons, or pathologically during disease. The dual leucine zipper kinase (DLK)/c-Jun--terminal kinase (JNK) pathway represents a conserved regulator of neuronal injury signaling that drives both neurodegeneration and axon regeneration, yet little is known about the factors that initiate DLK activity. Here, we uncover a novel role for a subfamily of MAP4 kinases consisting of MAP4K4, Traf2- and Nck-interacting kinase (TNIK or MAP4K7), and misshapen-like kinase 1 (MINK1 or MAP4K6) in regulating DLK/JNK signaling in neurons. Inhibition of these MAP4Ks blocks stress-induced retrograde JNK signaling and protects from neurodegeneration, suggesting that these kinases may represent attractive therapeutic targets.

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