PDI-Mediated Reduction of Disulfide Bond on PSD95 Increases Spontaneous Seizure Activity by Regulating NR2A–PSD95 Interaction in Epileptic Rats Independent of S-Nitrosylation

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. N-terminal of the first two PDZ domains of the PSD95 recognizes a four amino acid consensus sequence (-E-S/T-D-V) and allows it to bivalently link various ion channels, including NR2 subunits [11][12][13]. PSD95 is also required for NMDAR-mediated neuronal nitric oxide synthase (nNOS) activation [14]. Indeed, suppression of PSD95 expression inhibits NMDAR-mediated NOS activation and neuronal excitotoxicity [14]. However, synaptic NMDAR currents, subunit expression, localization and synaptic morphology remain unaltered in transgenic mice lacking PSD95, but they also shift the frequency dependence of NMDAR-mediated synaptic plasticity and impair spatial learning [15]. Thus, it is emphasized that PSD95 is critical in coupling NMDAR to cellular signaling networks and plays an important role in their biological functions within the central nervous system.On the other hand, the N-terminus of PSD95 contains a pair of cysteine residue (cysteine 3 and 5 sites). These cysteine residues are potential sites for disulfide-bond formation, S-nitrosylation and/or palmitoylation [16,17]. In addition, disulfide linkage of each PSD95 protein in these sites mediates its multimerization as supramolecular aggregates [18] and facilitates receptor-clustering activity [19]. S-nitrosylation of these sites also influences PSD95 palmitoylation and its clustering at synapses [20]. 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. Here, 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. In additi...

PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

“…2 ). GluA2 S880 phosphorylation facilitates AMPAR internalization 18 , 19 , while GluA1 S831 phosphorylation is not required for AMPAR trafficking 26 , 27 . Therefore, our findings indicate that PDI siRNA may diminish surface AMPAR expression by increasing p-GluA2 S880 level under physiological condition.…”

“…These discrepancies would be consequences from properties of DTNB and PDI siRNA: DTNB oxidizes C residues on proteins exposed to the extracellular milieu, because of poor membrane permeability 17 . Unlike DTNB, PDI siRNA regulates the thiol modification of proteins in ER, cytosol and cell surface 6 , 10 , 18 , 19 . Furthermore, PDI serves as a nitric oxide (NO) donor causing S -nitrosylation of C residues that is a posttranslational protein modification 20 , 21 .…”

Redox-dependent synaptic clustering of gephyrin

“…Recently, the disruption of a cysteine linkage in PSD95 has been linked to an epileptic phenotype in rats 39 highlighting the importance of redox-regulation of neuronal scaffold proteins.…”

“…In comparison to PSD95, the excitatory pendant, multimerization by the formation of disulfide bridges was also detected in cellulo and in vivo 38 . Recently, the disruption of a cysteine linkage in PSD95 has been linked to an epileptic phenotype in rats 39 highlighting the importance of redox-regulation of neuronal scaffold proteins.…”

Behavioral and Molecular Responses to Exogenous Cannabinoids During Pentylenetetrazol-Induced Convulsions in Male and Female Rats

“…Furthermore, it is not known whether exogenous cannabinoids can mitigate the impact that chronic or acute seizures have on the levels of synaptic proteins that are important mediators of excitation, cognition, and/or neurotoxicity (e.g., postsynaptic density protein 95, PSD-95; GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, AMPAR; Ca 2+ /calmodulin-dependent protein kinase II, CaMKII) (Bronstein et al, 1990;Dong and Rosenberg, 2004;Ying et al, 2004;Needs et al, 2019;Lee and Kim, 2020). We therefore investigated whether similar changes could manifest early on, i.e., after an acute dose of PTZ, considered a ''gold standard'' in the evaluation of AEDs (Bialer and White, 2010), and whether underlying sex differences may be present.…”