Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability.

Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.

Soy-deficient diet induces renal lesions in juvenile rats.

Certified LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 IF (5K96) is a commercial diet low in soy isoflavones developed for use in developmental and reproductive toxicity (DART) studies, especially those designed to detect endocrine disruptors. The objective of this study was to determine the incidences and severities of 5K96-associated renal lesions in control F0 and F1 cohorts of rats fed the 5K96 diet. Kidneys from control animals of four DART studies involving Sprague-Dawley rats fed the 5K96 diet, were evaluated microscopically. Mineralization and basophilic tubules were present in high incidence/severity in males and females compared to historical controls fed conventional diets. F1 cohorts were affected to a far greater degree than F0 cohorts, and females were affected more than males. Consideration of target tissue and mode of action should be given before automatically incorporating the 5K96 diet into DART study designs, and caution should be exercised when identifying and interpreting renal toxicity in the F1 cohorts of such studies.

Neuroprotective Effects of AEOL10150 in a Rat Organophosphate Model.

Prolonged seizure activity or status epilepticus (SE) is one of the most critical manifestations of organophosphate exposure. Previous studies in our laboratory have demonstrated that oxidative stress is a critical mediator of SE-induced neuronal injury. The goal of this study was to determine if diisopropylflurorphoshate (DFP) exposure in rats resulted in oxidative stress and whether scavenging reactive oxygen species attenuated DFP-induced neurotoxicity. DFP treatment increased indices of oxidative stress in a time- and region- dependent manner. Neuronal loss measured by Fluoro-Jade B staining was significantly increased in the hippocampus, piriform cortex and amygdala following DFP. Similarly, levels of the proinflammatory cytokines, particularly TNF-α, IL-6, and KC/GRO were significantly increased in the piriform cortex and in the hippocampus following DFP treatment. The catalytic antioxidant AEOL10150, when treatment was initiated 5 min after DFP-induced SE, significantly attenuated indices of oxidative stress, neuroinflammation and neuronal damage. This study suggests that catalytic antioxidant treatment may be useful as a novel therapy to attenuate secondary neuronal injury following organophosphate exposure.

Scavenging reactive oxygen species inhibits status epilepticus-induced neuroinflammation.

Inflammation has been identified as an important mediator of seizures and epileptogenesis. Understanding the mechanisms underlying seizure-induced neuroinflammation could lead to the development of novel therapies for the epilepsies. Reactive oxygen species (ROS) are recognized as mediators of seizure-induced neuronal damage and are known to increase in models of epilepsies. ROS are also known to contribute to inflammation in several disease states. We hypothesized that ROS are key modulators of neuroinflammation i.e. pro-inflammatory cytokine production and microglial activation in acquired epilepsy. The role of ROS in modulating seizure-induced neuroinflammation was investigated in the pilocarpine model of temporal lobe epilepsy (TLE). Pilocarpine-induced status epilepticus (SE) resulted in a time-dependent increase in pro-inflammatory cytokine production in the hippocampus and piriform cortex. Scavenging ROS with a small-molecule catalytic antioxidant decreased SE-induced pro-inflammatory cytokine production and microglial activation, suggesting that ROS contribute to SE-induced neuroinflammation. Scavenging ROS also attenuated phosphorylation of ribosomal protein S6, the downstream target of the mammalian target of rapamycin (mTOR) pathway indicating that this pathway might provide one mechanistic link between SE-induced ROS production and inflammation. Together, these results demonstrate that ROS contribute to SE-induced cytokine production and antioxidant treatment may offer a novel approach to control neuroinflammation in epilepsy.

Hippocampal TNFα Signaling Contributes to Seizure Generation in an Infection-Induced Mouse Model of Limbic Epilepsy.

Central nervous system infection can induce epilepsy that is often refractory to established antiseizure drugs. Previous studies in the Theiler's murine encephalomyelitis virus (TMEV)-induced mouse model of limbic epilepsy have demonstrated the importance of inflammation, especially that mediated by tumor necrosis factor-α (TNFα), in the development of acute seizures. TNFα modulates glutamate receptor trafficking via TNF receptor 1 (TNFR1) to cause increased excitatory synaptic transmission. Therefore, we hypothesized that an increase in TNFα signaling after TMEV infection might contribute to acute seizures. We found a significant increase in both mRNA and protein levels of TNFα and the protein expression ratio of TNF receptors (TNFR1:TNFR2) in the hippocampus, a brain region most likely involved in seizure initiation, after TMEV infection, which suggests that TNFα signaling, predominantly through TNFR1, may contribute to limbic hyperexcitability. An increase in hippocampal cell-surface glutamate receptor expression was also observed during acute seizures. Although pharmacological inhibition of TNFR1-mediated signaling had no effect on acute seizures, several lines of genetically modified animals deficient in either TNFα or TNFRs had robust changes in seizure incidence and severity after TMEV infection. TNFR2-/- mice were highly susceptible to developing acute seizures, suggesting that TNFR2-mediated signaling may provide beneficial effects during the acute seizure period. Taken together, the present results suggest that inflammation in the hippocampus, caused predominantly by TNFα signaling, contributes to hyperexcitability and acute seizures after TMEV infection. Pharmacotherapies designed to suppress TNFR1-mediated or augment TNFR2-mediated effects of TNFα may provide antiseizure and disease-modifying effects after central nervous system infection.

Post-translational Activation of Glutamate Cysteine Ligase with Dimercaprol: A NOVEL MECHANISM OF INHIBITING NEUROINFLAMMATION IN VITRO.

Neuroinflammation and oxidative stress are hallmarks of various neurological diseases. However, whether and how the redox processes control neuroinflammation is incompletely understood. We hypothesized that increasing cellular glutathione (GSH) levels would inhibit neuroinflammation. A series of thiol compounds were identified to elevate cellular GSH levels by a novel approach (i.e. post-translational activation of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis). These small thiol-containing compounds were examined for their ability to increase intracellular GSH levels in a murine microglial cell line (BV2), of which dimercaprol (2,3-dimercapto-1-propanol (DMP)) was found to be the most effective compound. DMP increased GCL activity and decreased LPS-induced production of pro-inflammatory cytokines and inducible nitric-oxide synthase induction in BV2 cells in a concentration-dependent manner. The ability of DMP to elevate GSH levels and attenuate LPS-induced pro-inflammatory cytokine production was inhibited by buthionine sulfoximine, an inhibitor of GCL. DMP increased the expression of GCL holoenzyme without altering the expression of its subunits or Nrf2 target proteins (NQO1 and HO-1), suggesting a post-translational mechanism. DMP attenuated LPS-induced MAPK activation in BV2 cells, suggesting the MAPK pathway as the signaling mechanism underlying the effect of DMP. Finally, the ability of DMP to increase GSH via GCL activation was observed in mixed cerebrocortical cultures and N27 dopaminergic cells. Together, the data demonstrate a novel mechanism of GSH elevation by post-translational activation of GCL. Post-translational activation of GCL offers a novel targeted approach to control inflammation in chronic neuronal disorders associated with impaired adaptive responses.