GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor-associated epilepsy

Abstract

Seizures frequently accompany gliomas and often escalate to peritumoral epilepsy. Previous work revealed the importance of tumor-derived excitatory glutamate (Glu) release mediated by the cystine-glutamate transporter (SXC) in epileptogenesis. We now show a novel contribution of GABAergic disinhibition to disease pathophysiology. In a validated mouse glioma model, we found that peritumoral parvalbumin-positive GABAergic inhibitory interneurons are significantly reduced, corresponding with deficits in spontaneous and evoked inhibitory neurotransmission. Most remaining peritumoral neurons exhibit elevated intracellular Cl− concentration ([Cl−]i) and consequently depolarizing, excitatory GABA responses. In these neurons, the plasmalemmal expression of KCC2, which establishes the low [Cl−]i required for GABAAR-mediated inhibition, is significantly decreased. Interestingly, reductions in inhibition are independent of Glu release, but the presence of both decreased inhibition and decreased SXC expression is required for epileptogenesis. We suggest GABAergic disinhibition renders peritumoral neuronal networks hyper-excitable and susceptible to seizures triggered by excitatory stimuli, and propose KCC2 as a therapeutic target.

Animal models of tumour-associated epilepsy

“…Concurring with this, the density of the sodium-potassium-chloride co-transporter NKCC1 was increased in these membrane fragments (Conti et al, 2011). A recent paper by Campbell et al (2015) more directly supports this: using a primary cell human glioma tumour implant, peritumoural neurons show a more depolarised E GABA , an increased NKCC expression as well as a decreased expression of chloride uptake transporter KCC2, incidentally also correlating with patient survival (Campbell et al, 2015). Finally, also changes in intrinsic firing properties (increased incidence of bursting neurons) may play a role, as already indicated in human glioma tissue (Williamson et al, 2003), and also seen in a C6-glioma cell line model , as perhaps also loss of protective mechanisms, since adenosine degradation via adenosine kinase has been demonstrated to be increased in peritumoural tissue, and especially in astrocytomas, where adenosine kinase is significantly overexpressed (de Groot et al, 2012).…”

“…"clinical signs". The reason for this may be that the authors (often concentrating on oncological aspects) did not look for such signs, and indeed overlooked them since in many cases, even if seizure-like activity is being observed in the EEG, behavioural signs are often quite subtle, comprising freezing, facial automatisms and head tremor, or massive startle response in reaction to audiogenic stimuli, and only very seldom generalised tonic-clonic convulsions (Buckingham et al, 2011;Campbell et al, 2015;Köhling et al, 2006). Another reason may be that tumour cell injection into areas other than the neocortex such as capsula interna, corpus striatum or even cerebellar subdural space, are probably not ideal for the purpose of an epilepsy model (Aas et al, 1995;Beaumont et al, 1996;Hossmann et al, 1989;Krajewski et al, 1986;Linn et al, 1989;Rewers et al, 1990;Wechsler et al, 1989).…”

“…5% methylcellulose, 10 l), usually with a total cell number of 2.5 × 10 5 to 2 × 10 6 . This results in tumour growth within 7-15 days to a size of 1-2 mm in diameter, and in the case of neocortically injected tumour cells, in seizures, or at least EEG abnormalities and in vitro hyperexcitability, including spontaneous discharges (Buckingham et al, 2011;Campbell et al, 2012Campbell et al, , 2015Köhling et al, 2006). As it can be inferred form Table 1, one of the main problems of such models appears to be that in most cases, seizures are not being reported, even cursorily under the heading of e.g.…”

“…Thus, they either rely on human cell lines (Campbell et al, 2012), human primary cell cultures maintained in nude, i.e. immunocompromized, mice (Buckingham et al, 2011;Campbell et al, 2015), or on a rat cell Q5 line Senner et al, 2004), with mixed cell lines being used in some cases (Beaumont et al, 1996). Cell lines have the advantage of a relatively easy cultivation under cell culture conditions in appropriate media such as DMEM/F12 (Invitrogen) supplemented with 7% bovine growth serum and glutamine (Campbell et al, 2012), or DMEM supplemented with 10% fetal calf serum and antibiotics .…”

“…in vitro slice experiments, enabling the experimenter to delineate the tumour mass or even identify single glioma cells. Primary cell cultures, in turn, necessitate more effort in cultivation, as they need to be maintained in serial passage in immunodeficient (athymic nude) mice, and are then harvested for transplant, being intermittently kept in more expensive media such as Neurobasal A medium (Invitrogen) supplemented with epidermal and fibroblast growth factors, B-27 supplement without vitamin A (Invitrogen) as well as antibiotics (Campbell et al, 2015;Buckingham et al, 2011). Another disadvantage is that stable GFP-labelling is not possible, and that they apparently are working mainly in immunodeficient (scid) mice only.…”