Abstract
Redox modulation of cysteine residues is one of the post-translational modifications of N-methyl-D-aspartate receptor (NMDAR). Protein disulfide isomerases (PDI), an endoplasmic reticulum (ER) chaperone, plays a crucial role in catalyzing disulfide bond formation, reduction, and isomerization. In the present study, we found that PDI bound to NMDAR in the normal hippocampus, and that this binding was increased in chronic epileptic rats. In vitro thiol reductase assay revealed that PDI increased the amount of thiols on full-length recombinant NR1 protein. PDI siRNA, 5–5′-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin and PDI antibody reduced seizure susceptibility in response to pilocarpine. In addition, PDI knockdown effectively ameliorated spontaneous seizure activity in chronic epileptic rats. Anticonvulsive effects of PDI siRNA were correlated to the reduction of the amount of free- and nitrosothiols on NMDAR, accompanied by the inhibition of PDI activity. However, PDI knockdown did not lead to alteration in basal neurotransmission or ER stress under physiological condition. These findings provide mechanistic insight into sulfhydration of disulfide bonds on NMDAR by PDI, and suggest that PDI may represent a target of potential therapeutics for epilepsy, which avoids a possible side effect on physiological receptor functionality.
Highlights
Redox modulation of cysteine residues is one of the post-translational modifications of N-methylD-aspartate receptor (NMDAR)
To elucidate whether the transient up-regulation of Protein disulfide isomerases (PDI) overexpression plays an anti-convulsive or pro-convulsive role, we investigated the effect of PDI knockdown on seizure susceptibility in response to PILO
To confirm whether cell-surface or secreted PDI is involved in NMDAR-mediated neuronal excitability, we investigated the presence of PDI in cerebrospinal fluid (CSF) and the effect of PDI neutralization by a specific PDI antibody on seizure susceptibility in response to PILO
Summary
Redox modulation of cysteine residues is one of the post-translational modifications of N-methylD-aspartate receptor (NMDAR). PDI knockdown did not lead to alteration in basal neurotransmission or ER stress under physiological condition These findings provide mechanistic insight into sulfhydration of disulfide bonds on NMDAR by PDI, and suggest that PDI may represent a target of potential therapeutics for epilepsy, which avoids a possible side effect on physiological receptor functionality. We have reported that PDI expression in dentate granule cells was transiently increased following status epilepticus (SE, a prolonged seizure activity) induced by pilocarpine (PILO), and restored to basal level at 4 weeks after SE23 With respect to these profiles of PDI, we hypothesized that PDI may be one of endogenous reducing factors for the disulfide bonds of NMDAR redox sites, and the consequent potentiation of NMDAR function could increase seizure susceptibility and sustain seizure activity. Our findings suggest that PDI may involve the dynamic redox regulation of NMDAR function, which is a critical mechanism in the seizure susceptibility, and may represent a target of potential therapeutics for epilepsy
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