Abstract
Postsynaptic density-95 (PSD95), a major scaffolding protein, is critical in coupling N-methyl-D-aspartate receptor (NMDAR) to cellular signaling networks in the central nervous system. A couple of cysteine residues in the N-terminus of PSD95 are potential sites for disulfide bonding, S-nitrosylation and/or palmitoylation. Protein disulfide isomerase (PDI) reduces disulfide bonds (S-S) to free thiol (-SH) on various proteins. However, the involvement of PDI in disulfide bond formation/S-nitrosylation of PSD95 and its role in epilepsy are still unknown. In the present study, acute seizure activity significantly increased the bindings of PDI to NR2A, but not to PSD95, while it decreased the NR2A–PSD95 binding. In addition, pilocarpine-induced seizures increased the amount of nitrosylated (SNO-) thiols, not total (free and SNO-) thiols, on PSD95. Unlike acute seizure, spontaneous seizing rats showed the increases in PDI–PSD95 binding, total- and SNO-thiol levels on PSD95, and NR2A–PSD95 interaction. PDI siRNA effectively reduced spontaneous seizure activity with decreases in total thiol level on PSD95 and NR2A–PSD95 association. These findings indicate that PDI-mediated reduction of disulfide-bond formations may facilitate the NR2A–PSD95 binding and contribute to spontaneous seizure generation in epileptic animals.
Highlights
The N-methyl-D-aspartate receptor (NMDAR) is one of the major excitatory receptors contributing to neurotransmission, synaptic plasticity and neuronal damage [1,2,3]
Since NR2A regulates channel open probability and drives synaptic strength [32,33], our findings indicate that the reduced NR2A–Postsynaptic density-95 (PSD95) interaction in acute seizure model may be an adaptive response for inhibiting excessive neuronal activity accompanied by the downregulation of both proteins
Our findings provide the possibility that protein disulfide isomerase (PDI)-mediated reduction of disulfide bonds on PSD95 may increase NR2A–PSD95 binding and lead to spontaneous seizure activity via NMDAR hyperactivation in epileptic animals, concomitant with PDI-mediated reduction of NR2A [5]
Summary
The N-methyl-D-aspartate receptor (NMDAR) is one of the major excitatory receptors contributing to neurotransmission, synaptic plasticity and neuronal damage [1,2,3]. Suppression of PSD95 expression inhibits NMDAR-mediated NOS activation and neuronal excitotoxicity [14]. Given the reductive role of PDI in disulfide bonds, it is interesting to evaluate whether PDI mediates disulfide-bond formation or S-nitrosylation on PSD95 during seizure generation. We demonstrate that PILO-induced acute seizure activity significantly increased the bindings of PDI to NR2A, but not to PSD95, while it decreased the NR2A–PSD95. Spontaneously epileptic rats showed the increases in PDI–PSD95 binding, total- and SNO-thiol levels on PSD95, and NR2A–PSD95 interaction. PDI siRNA effectively reduced spontaneous seizure activity with the decreases in the total thiol level on PSD95 and NR2A–PSD95 association. We suggest that PDI-mediated PSD95 redox may increase seizure susceptibility via facilitation of NR2A–PSD95 binding in epileptic animals
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