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.

Pharmacological elevation of glutathione inhibits status epilepticus-induced neuroinflammation and oxidative injury.

Glutathione (GSH) is a major endogenous antioxidant, and its depletion has been observed in several brain diseases including epilepsy. Previous studies in our laboratory have shown that dimercaprol (DMP) can elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme and inhibit neuroinflammation in vitro. Here we determined 1) the role of cysteamine as a new mechanism by which DMP increases GSH biosynthesis and 2) its ability to inhibit neuroinflammation and neuronal injury in the rat kainate model of epilepsy. DMP depleted cysteamine in a time- and concentration-dependent manner in a cell free system. To guide the in vivo administration of DMP, its pharmacokinetic profile was determined in the plasma, liver, and brain. The results confirmed DMP's ability to cross the blood-brain-barrier. Treatment of rats with DMP (30 mg/kg) depleted cysteamine in the liver and hippocampus that was associated with increased GCL activity in these tissues. GSH levels were significantly increased (20 %) in the hippocampus 1 h after 30 mg/kg DMP administration. Following DMP (30 mg/kg) administration once daily, a marked attenuation of GSH depletion was seen in the SE model. SE-induced inflammatory markers including cytokine release, microglial activation, and neuronal death were significantly attenuated in the hippocampus with DMP treatment. Taken together, these results highlight the importance of restoring redox status with rescue of GSH depletion by DMP in post epileptogenic insults.

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.