Anomalous levels of Cl- transporters cause a decrease of GABAergic inhibition in human peritumoral epileptic cortex

Purpose: Several factors contribute to epileptogenesis in patients with brain tumors, including reduced gamma-aminobutyric acid (GABA) ergic inhibition. In particular, changes in Cl- homeostasis in peritumoral microenvironment, together with alterations of metabolism, are key processes leading to epileptogenesis in patients afflicted by glioma. It has been recently proposed that alterations of Cl- homeostasis could be involved in tumor cell migration and metastasis formation. In neurons, the regulation of intracellular Cl- concentration ([Cl-](i)) is mediated by NKCC1 and KCC2 transporters: NKCC1 increases while KCC2 decreases [Cl-] i. Experiments were thus designed to investigate whether, in human epileptic peritumoral cortex, alterations in the balance of NKCC1 and KCC2 activity may decrease the hyperpolarizing effects of GABA, thereby contributing to epileptogenesis in human brain tumors. Methods: Membranes from peritumoral cortical tissues of epileptic patients afflicted by gliomas (from II to IV WHO grade) and from cortical tissues of nonepileptic patients were injected into Xenopus oocytes leading to the incorporation of functional GABAA receptors. The GABA-evoked currents were recorded using standard two-microelectrode voltage-clamp technique. In addition, immunoblot analysis and immunohistochemical staining were carried out on membranes and tissues from the same patients. Key Findings: We found that in oocytes injected with epileptic peritumoral cerebral cortex, the GABA-evoked currents had a more depolarized reversal potential (E-GABA) compared to those from nonepileptic healthy cortex. This difference of EGABA was abolished by the NKCC1 blocker bumetanide or unblocking of KCC2 with the Zn2+ chelator TPEN. Moreover, Western blot analysis revealed an increased expression of NKCC1, and more modestly, of KCC2 transporters in epileptic peritumoral tissues compared to nonepileptic control tissues. In addition, NKCC1 immunoreactivity was strongly increased in peritumoral cortex with respect to nonepileptic cortex, with a prominent expression in neuronal cells. Significance: We report that the positive shift of EGABA in epileptic peritumoral human cortex is due to an altered expression of NKCC1 and KCC2, perturbing Cl- homeostasis, which might lead to a consequent reduction in GABAergic inhibition. These findings point to a key role of Cl- transporters KCC2 and NKCC1 in tumor-related epilepsy, suggesting a more specific drug therapy and surgical approaches for the epileptic patients afflicted by brain tumors.