Abstracts

Rationale:
Neuroinflammation, network hyperexcitability and dysregulated glutathione (GSH) redox status are all detrimental epileptogenic factors that can perpetuate seizures. Treatment strategies that increased intracellular GSH levels have shown to decrease proinflammatory cytokine release (McElroy, PB et al., 2017) and reduce seizure frequency (Pauletti, A et al., 2019). However, a burning question remains: how does GSH redox modulation control neuroinflammation and neuronal hyperexcitability? Interestingly, the redox-sensitive mammalian target of rapamycin (mTOR) pathway is dysregulated in several genetic and acquired epilepsy models . Since mTOR is a ‘master regulator’ of critical processes like neurotransmission, and inflammatory responses, we asked whether GSH-mediated redox modulation of mTOR controls neuroinflammation and neuronal hyperexcitability in vitro.
Method:
Rat primary neuronal-glial cultures were treated with compounds that could modulate intracellular GSH levels and then stimulated them with a convulsant. Endpoints such as neuronal hyperexcitability, intracellular GSH levels, target protein level alterations and redox modifications were assessed.
Results:
We have previously shown that 2,3-dimercapto-1-propanol (DMP), a thiol-containing drug significantly increases intracellular GSH levels in rat primary neuronal-glial cultures and murine microglial cell line (BV2) by a novel mechanism: post-translational activation of the rate-limiting enzyme in GSH biosynthesis. We showed that elevating GSH levels using DMP attenuates lipopolysaccharide-induced proinflammatory cytokine production in BV2 cells (McElroy, PB et al., 2017). In the current study, we determined if increased GSH levels would attenuate ‘seizure-like’ activity and whether the redox-sensitive mTOR pathway mediated this effect in vitro. Pre-treatment of neuronal-glial cultures with 30µM or 100µM DMP for 4h increased GSH levels (p< 0.001 vs vehicle control (VC)) and significantly decreased 4-aminopyridine (4AP, voltage-gated potassium channel blocker) induced neuronal hyperexcitability (p< 0.01, p< 0.0001 vs 1mM 4AP). Incubation of cultures with 1mM 4AP for 4h increased mTORC1 activity (p< 0.01 vs VC) (assessed by pS6/S6 levels) which was inhibited by pre-treatment with 30µM DMP for 4h (p< 0.01 vs 1mM 4AP). Next, cultures in which GSH was depleted by buthionine sulfoximine (BSO) for 24h showed aberrant activation of  mTORC1 in a dose-dependent manner (p< 0.05 vs VC). Surprisingly, cultures pre-treated with 30µM DMP for 4h and then with 100µM BSO for 20h were protected against mTORC1 hyperactivation. Assessment of redox modifications in cultures treated with 300µM BSO for 24h revealed >