Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

Abstract

Context: Abnormality in interactions between N-methyl-d-aspartate (NMDA) receptor and its signaling molecules occurs in the lesioned striatum in Parkinson's disease (PD) and levodopa-induced dyskinesia (LID). It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95), one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated.

Methods: In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily) intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO) on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation.

Results: Levodopa administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the lesioned striatum of PD rats. However, the protein levels of Fyn and NR2B showed no difference under the above conditions.

Conclusion: The data demonstrate that the inhibition of PSD-95 protein expression suppressed the interactions of Fyn with NR2B and NR2B tyrosine phosphorylation and subsequently downregulated NMDA receptor overactivation, thus providing benefit for the therapy of LID. Therefore, PSD-95 is important for overactivity of NMDA receptor function due to facilitating NR2B tyrosine phosphorylation dependent on Fyn kinase by regulating interactions of Fyn with NR2B under the pathological conditions of LID.

Keywords: NMDA; PSD-95 ASO; rotational response.

Figure 1

Effect of intrastriatally administered PSD-95 ASO on rotational duration (A) and peak rotations (B) of rotational response to levodopa. Notes: Rotational duration and peak rotations were compared on days 1 and 22 of levodopa treatment and 3 days after withdrawal of levodopa treatment + PSD-95 ASO or PSD-95 MSO. At 3 days after levodopa withdrawal, either PSD-95 ASO or PSD-95 MSO was administered before acute levodopa challenge. *P<0.05 versus day 1; ^#P<0.05 versus day 22; ^&P<0.05 versus PSD-95 ASO group on day 22. Abbreviations: PSD-95 ASO, PSD-95mRNA antisense oligonucleotides; PSD-95 MSO, PSD-95mRNA scrambled missense oligonucleotides.

Figure 2

Effects of PSD-95 ASO on the expression of PSD-95 in lesioned rat striatum. Notes: Samples were from the group of control, PD, and chronic L-dopa treatment rats and after withdrawal of L-dopa treatment + PSD-95 ASO or PSD-95 MSO (n=5). (A) IB analysis of the protein levels of PSD-95 with anti-PSD-95 antibody. (B) Quantitative representation of the protein levels of PSD-95. *P<0.05 versus control; ^#P<0.05 versus PD; ^&P<0.05 versus chronic L-dopa (day 22) group; ^$P<0.05 versus PSD-95 ASO group. A significant attenuation of PSD-95 levels was observed in the PSD-95 ASO-injected striatum. The results were repeatable on striatal extracts from five rats. Abbreviations: IB, immunoblotting; L-dopa, levodopa; PD, Parkinson’s disease; PSD-95 ASO, PSD-95mRNA antisense oligonucleotides; PSD-95 MSO, PSD-95mRNA scrambled missense oligonucleotides.

Figure 3

Effects of pretreatment with PSD-95 ASO or MSO on the augmented tyrosine phosphorylation of NR2B induced by chronic L-dopa treatment in lesioned rat striatum. Notes: Samples were from the group of control, PD, and chronic L-dopa treatment rats and L-dopa treatment + PSD-95 ASO or PSD-95 MSO (n=5). (A) Tyrosine phosphorylation of NR2B examined by IP with anti-NR2B antibody followed by IB with anti-pY. (B) IB analysis of the protein levels of NR2B with anti-NR2B antibody. (C) Quantitative representation of NR2B tyrosine phosphorylation (p-NR2B) and the protein levels of NR2B. ^#P<0.05 versus PD; ^&P<0.05 versus chronic L-dopa (day 22) group; ^$P<0.05 versus PSD-95 ASO group. The results were repeatable on striatal extracts from five rats. Abbreviations: anti-pY, antibody against phosphotyrosine; IB, immunoblotting; IP, immunoprecipitation; L-dopa, levodopa; PD, Parkinson’s disease; PSD-95 ASO, PSD-95mRNA antisense oligonucleotides; PSD-95 MSO, PSD-95mRNA scrambled missense oligonucleotides.

Figure 4

Effects of pretreatment with PSD-95 ASO or MSO on the augmented interactions of Fyn with NR2B induced by chronic L-dopa treatment in lesioned rat striatum. Notes: Samples were from the group of control, PD, chronic L-dopa treatment rats and after withdrawal of L-dopa treatment + PSD-95 ASO or PSD-95 MSO (n=5). (A) Co-immunoprecipitation analysis of interactions of Fyn with NR2B. Sample proteins were immunoprecipitated with anti-Fyn antibodies and then blotted (IB) with anti-NR2B antibody. (B) IB analysis of the protein levels of Fyn with anti-Fyn antibodies. (C) Quantitative representation of interactions of Fyn with NR2B. *P<0.05 versus control; ^#P<0.05 versus PD; ^&P<0.05 versus chronic L-dopa (day 22) group; ^$P<0.05 versus PSD-95 ASO group. The results were repeatable on striatal extracts from five rats. Abbreviations: IB, immunoblotting; IP, immunoprecipitation; L-dopa, levodopa; PD, Parkinson’s disease; PSD-95 ASO, PSD-95mRNA antisense oligonucleotides; PSD-95 MSO, PSD-95mRNA scrambled missense oligonucleotides.