Decreased epileptogenesis in mice lacking the System x_c⁻ transporter occurs in association with a reduction in AMPA receptor subunit GluA1

Abstract

Summary Objective Although the cystine/glutamate antiporter System xc− (Sxc−) plays a permissive role in glioma‐associated seizures, its contribution to other acquired epilepsies has not been determined. As such, the present study investigates whether and how Sxc− contributes to the pentylenetetrazole (PTZ) chemical kindling model of epileptogenesis. Methods Male Sxc− null (sut/sut) mice and their wild‐type littermates were administered PTZ (i.p.) daily for up to 21 days (kindling paradigm). Seizure severity was scored on a 5‐point behavioral scale. Mossy fiber sprouting, cellular degeneration, and Sxc− light chain (xCT) messenger RNA (mRNA) were explored using Timm staining, thionin staining, and real‐time quantitative polymerase chain reaction (qPCR), respectively. Levels of reduced and oxidized glutathione and cysteine were determined via high‐performance liquid chromatography (HPLC). Plasma membrane protein levels of glutamate and γ‐aminobutyric acid (GABA) receptor subunits as well as the K+/Cl− co‐transporter KCC2 were quantified via western blot analysis. Results Repeated administration of PTZ produced chemical kindling in only 50% of Sxc− null mice as compared to 82% of wild‐type littermate control mice. Kindling did not result in any changes in xCT mRNA levels assessed in wild‐type mice. No cellular degeneration or mossy fiber sprouting was discernible in either genotype. Except for a small, but significant, decrease in oxidized cysteine in the hippocampus, no other change in measured redox couples was determined in Sxc− null mice. Cortical levels of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subunit GluA1 were decreased in Sxc− null mice as compared to wild‐type littermates, whereas all other proteins tested showed no difference between genotypes. Significance This study provides the first evidence that Sxc− signaling contributes to epileptogenesis in the PTZ kindling model of acquired epilepsy. Further data indicate that a reduction in AMPA receptor signaling could underlie the resistance to PTZ kindling uncovered in Sxc− null mice.

Hyperexcitability and brain morphological differences in mice lacking the cystine/glutamate antiporter, system xc−

“…However, it may be accounted for by differences in how the Sx c − null was created (natural mutation (Chintala et al, 2005) vs. targeted deletion (Sato et al, 2005)), the mouse strain (C3H/HeSnJ vs. C57BL/6) (Loscher et al, 2017), or by the chemoconvulsants used, as well as their method of administration. Finally, these acute studies are different from previous work by ourselves and others demonstrating that loss of Sx c − signaling in male mice-either via mutation, targeted deletion, or pharmacological block-reduced epileptogenesis (i.e., the process of acquiring epilepsy) in several different electrical and chemoconvulsant models (Leclercq et al, 2019;Sears et al, 2019).…”

Tonic extracellular glutamate and ischaemia: glutamate antiporter system xc− regulates anoxic depolarization in hippocampus

“…Finally, blockade of glutamate receptors eliminated the differences in AD latency between xCT −/− and WT mice, suggesting that the lower levels of tonic glutamate in xCT −/− mice delay AD by slowing the activation of postsynaptic glutamate receptors during anoxia. These results reinforce, extend, and interpret findings of reduced neuronal death after ischaemic challenge in cell culture systems (Hsieh et al, 2017;Jackman et al, 2012;Soria et al, 2014;Thorn et al, 2015) as well as protection from epileptogenesis (De Bundel et al, 2011;Leclercq et al, 2019;Sears et al, 2019), cerebral ischaemia (Hsieh et al, 2017) and spinal cord injury (Sprimont et al, 2021) in vivo when system x c − is absent or inhibited.…”

The Cystine/Glutamate Antiporter, System xc–, Contributes to Cortical Infarction After Moderate but Not Severe Focal Cerebral Ischemia in Mice

“…Both of these findings might seem surprising given the importance of Sx c – to the production of GSH, at least, in vitro , where growth of xCT-deficient cells is dependent on the addition of a reducing agent ( Bannai and Tateishi, 1986 ; Chintala et al, 2005 ; Shih et al, 2006 ; Jackman et al, 2010 ). However, xCT –/– and Slc7a11 sut/sut mice have normal brain GSH levels ( De Bundel et al, 2011 ; Sears et al, 2019 ), at least under basal conditions, suggesting that other cyste(i)ne transporter systems compensate for loss in vivo ( Sosnoski et al, 2020 ). We cannot discount that there might be GSH dysregulation following ischemic stress.…”

“…At weaning, genotyping was performed via PCR analysis of tail genomic DNA samples as described ( Sears et al, 2019 ), after which mice were housed three to five per cage such that at least one mouse of each genotype was represented (pseudo-randomized design). Genotype was reconfirmed via PCR upon sacrifice.…”