Abstract
The intracellular concentration of chloride ([Cl-]i) determines the strength and polarity of GABA neurotransmission. STE20/SPS1-related proline/alanine-rich kinase (SPAK) is known as an indirect regulator of [Cl-]i for its activation of Na-K-2 Cl-co-transporters (NKCC) and inhibition of K-Cl-co-transporters (KCC) in many organs. NKCC1 or KCC2 expression changes have been demonstrated previously in the hippocampal neurons of mice with pilocarpine-induced status epilepticus (PISE). However, it remains unclear whether SPAK modulates [Cl-]i via NKCC1 or KCC2 in the brain. Also, there are no data clearly characterizing SPAK expression in cortical or hippocampal neurons or confirming an association between SPAK and epilepsy. In the present study, we examined SPAK expression and co-expression with NKCC1 and KCC2 in the hippocampal neurons of mice with PISE, and we investigated alterations in SPAK expression in the hippocampus of such mice. Significant increases in SPAK mRNA and protein levels were detected during various stages of PISE in the PISE mice in comparison to levels in age-matched sham (control) and blank treatment (control) mice. SPAK and NKCC1 expression increased in vitro, while KCC2 was down-regulated in hippocampal neurons following hypoxic conditioning. However, SPAK overexpression did not influence the expression levels of NKCC1 or KCC2. Using co-immunoprecipitation, we determined that the intensity of interaction between SPAK and NKCC1 and between SPAK and KCC2 increased markedly after oxygen-deprivation, whereas SPAK overexpression strengthened the relationships. The [Cl-]i of hippocampal neurons changed in a corresponding manner under the different conditions. Our data suggests that SPAK is involved in the plasticity of GABA signaling function in acquired epilepsy via adjustment of [Cl-]i in hippocampal neurons.
Highlights
Mesial temporal lobe epilepsy (MTLE) is recognized as one of the most medically intractable forms of epilepsy
pilocarpineinduced status epilepticus (PISE) was successfully established in about 76% of animals, the other mice died of violent convulsions
The up-regulation of SPS1-related proline/alanine-rich kinase (SPAK) at both the gene and protein level was examined in the hippocampus at various stages following induction of PISE in mice
Summary
Mesial temporal lobe epilepsy (MTLE) is recognized as one of the most medically intractable forms of epilepsy. One of the most important factors contributing to epileptogenesis is the persistent increase in intracellular chloride concentration ([Cl-]i), which induces a long-lasting shift in the action of g-aminobutyric acid (GABA) in the direction of depolarizing, leading to seizure generation [2]. In the immature brain or under pathological conditions, GABA exerts a depolarizing excitatory effect due to excessive intracellular accumulation of Cl-. Under such conditions, drugs such as benzodiazepines and phenobarbital exhibit reduced efficacy because GABA receptors are binding sites for these drugs [4]. It was discovered that chloride homeostasis can be regulated by several factors, including endogenous modulators [5]. Chloride homeostasis has become an attractive target for the treatment of central nervous system (CNS) disorders
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