Research reportTat-Src reduced NR2B tyrosine phosphorylation and its interaction with NR2B in levodopa-induced dyskinetic rats model
Introduction
Levodopa (L-dopa)-induced dyskinesia is regarded as the primary undesirable consequence of the dopaminergic drug clinically utilized in Parkinson’s disease (PD) therapy [1,2]. Even though the discovery of L-dopa set up an important landmark in the current neuropharmacology [3], the honeymoon period of L-dopa therapy is followed by the emergence of dyskinesia and wearing-off [1]. Yet, the potential pathogenesis of dyskinesia is still unclear.
The non-dopaminergic (eg, glutamatergic) mechanisms caused by the aberant dopaminergic stimulation are believed to be associated with the development of dyskinesia [[4], [5], [6]]. At the molecular level, the appearance of abnormal involuntary movements (AIMs) after chronic L-dopa therapy involves an abnormal form of N-methyl-D-aspartate receptor (NMDAR) property. Consequently, the phosphorylation state of NMDAR is viewed as the chief responsible for the property changes recently demonstrated in the striatum of dyskinetic rats [[7], [8], [9], [10]]. These alterations are characterized by the increased NMDAR subunit NR2B tyrosine phosphorylation level. Indeed, at this point, our previous research had demonstrated that chronic L-dopa therapy lead to the increased NR2B tyrosine phosphorylation depending on the associations of NR2B with Src family protein tyrosine kinase [11,12].
Src is one protein tyrosine kinase and widely expressed in the striatum. As an important regulating kinase on NMDAR function, Src functionally colocalized in the postsynaptic membrane with NMDAR [13]. Although Fyn has been suggested to be the tyrosine kinase responsible for the phosphorylation of NR2B [[14], [15], [16], [17]], NR2B phosphorylation is not abolished in Fyn knockout mice. This suggests that Src family tyrosine kinases other than Fyn can also phosphorylate NR2B. Indeed, NR2B phosphorylation is modulated by cycline-dependent kinase lV (Cdk4) and PSD-95 in a Src-dependent but Fyn-independent manner [18]. Especially, Src can combine with NR2B by its Src-homology 2 (SH2) domain. The association between Src and NR2B is capable of activating Src tyrosine kinase, which facilitates NR2B tyrosine phosphorylation and enhances NMDAR function [[19], [20], [21], [22]]. The activation of NMDAR can conversely increase the activity of Src due to Ca2+ influx mediated by NMDAR [[23], [24], [25]]. Thus, this cycle, if out of control, could lead to overactivation of both NMDAR and Src.
One Src-derived interfering peptide, Tat-Src (40–58) linked to the cell-permeable Tat motif, and previously demonstrated to prevent the association of Src with the NR2B subunit of NMDAR [26,27]. Considering the above mentioned evidences about Src and NR2B, in the present study, we aimed to investigate the effects of uncoupling the direct protein–protein interaction between Src and NR2B using the Src-derived interfering peptide (Tat-Src) in dyskinetic rats model.
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Animals
Adult female Sprague–Dawley rats (n = 60, Shanghai SIPPR-BK Laboratory Animal Company) weighing 200–250 g were used. Suitable living conditions and food and water were given to the rats. The Local Ethics Committee approved the procedures, which were conducted in accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals (NIH publication No. 80-23) and the Animals Research: Reporting In Vivo Experiments (ARRIVE) guidelines. According to the
Effects of Tat-Src on abnormal involuntary movements (AIMs)
As previously reported, chronic L-dopa treatment really give rise to abnormal involuntary movements in PD rats. Our data displayed that total AIMs scores showed a gradual upward trend in the process of L-dopa treatment in PD rats. On day 23, pretreatment of Tat-Src significantly alleviated the total AIMs scores. The total AIMs scores on day 23 averaged 14.8 ± 0.62 in dyskinesia group in comparison with 11.3 ± 0.51 in rats with Tat-Src-treated group (P < 0.05).
Similarly, the individual
Discussion
The present study explored the effect of pretreatment of Tat-Src on AIMs and neurochemical alterations in dyskinetic rats. Firstly, we displayed that pretreatment of Tat-Src can attenuate generally acknowledged dyskinetic scores in dyskinetic rats. Then, we confirmed the effectiveness of Tat-Src by demonstrating that pretreatment of Tat-Src could reduce the association of Src with NR2B without influencing the level of Src and NR2B. Meanwhile, we found that pretreatment of Tat-Src can
Conclusion
Taken together, our studies offer evidences that uncoupling of Src-NR2B association by the interfering peptide can reduce dyskinesia by downregulating NMDA function. The findings indicate that a manipulation of NMDAR signal complex by applying pharmacological agents characterized by a high specificity in reducing Src-NR2B formation can represent an important target of therapies to attentuate dyskinesia in PD patients. In addition, considering the role of Fyn in the regulation of NMDAR signal
Conflicting interests
There are no conflicting interests in the present research.
Funding
The present research was sponsored byChinese National Natural Science Foundation (No.81571234), Key research and development plan of Shandong Province (2018GSF118235), Shandong Natural Science Foundation (ZR2010HM117) and Yantai Science and Technology Development Project (2016WS037).
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Maowen Ba and Guoping Yu contributed equally to this work.