Research reportNR2B antagonist CP-101,606 inhibits NR2B phosphorylation at tyrosine-1472 and its interactions with Fyn in levodopa-induced dyskinesia rat model
Introduction
Levodopa (l-dopa) is the first-line drugs in the pharmacotherapy of Parkinson's disease (PD) [1]. However, l-dopa-induced dyskinesia (LID) is the major motor complication of long-term l-dopa treatment and has restricted l-dopa use for PD treatment [2], [3]. In the hemi-parkinsonian rat model by 6-hydroxydopamine (6-OHDA) lesion, l-dopa results in equal side effects named abnormal involuntary movements (AIMs). These adverse motor response alterations seem to be due to the pulsatile dopaminergic receptors activating on striatal neural cells, which results in reactive alterations in responding to cortex glutamate system input [4], [5].
Much research shows that the emergence of dyskinesia is related to reactive alterations in striatal neural cells that include overactivation of signal kinases connecting dopaminergic receptors to nearby glutamatergic receptors. N-Methyl-d-aspartate receptors (NMDAR) phosphorylated conditions experience alterations that obviously increase the force of cortex input to the striatal neural cells [5], [6], [7]. Moreover, the reactive alterations to pulsatile dopaminergic receptors activating seem to rely on NMDAR function enhancement since NMDAR antagonists improve dyskinesia in LID rat model [8], [9], [10]. Therefore, NMDAR appear to be responsible for mediating the emergence of LID.
NMDAR are heteromeric assemblies of receptor subunits (NR) and play an important role in synaptic plasticity [11], [12]. There are high levels of NR2B subunit expression throughout the striatum. NR2B tyrosine phosphorylations play an important role in the regulation of NMDAR function and have been connected to alterations of synaptic efficacy of NMDAR [13], [14]. Evidence showed that the augmented tyrosine phosphorylations of NR2B subunit result in the obvious increase in striatal NMDAR sensitivity and synaptic plasticity changes in dopaminergic responses in dyskinetic rats. The evidence also shows that chronic dopaminergic treatment leads to the persistent augment of striatal NMDAR response by enhanced NR2B tyrosine phosphorylation dependent on Fyn kinase, one Src family protein tyrosine kinase member [7], [15], [16]. In agreement with these findings, NR2B antagonist such as CP-101,606, has been demonstrated to improve dyskinesia in LID rat models [17]. Yet, the more accurate action mechanism by which CP-101,606 acts as one antidyskinetic reagent is less well understood.
Under normal circumstances, Fyn may bind with NR2B through its Src-homology 2 (SH2) domain. The research shows that the association between NR2B and Fyn can activate Src family protein tyrosine kinase, which promotes NR2B tyrosine phosphorylation [11], [12], [13], [14]. In view of the above relationship between NR2B and Fyn, we speculate that the altered association of NR2B with Fyn may be involved in l-dopa-induced enhancement of NMDAR function by facilitating NR2B tyrosine phosphorylation dependent on Fyn kinase in dyskinetic rat models. In addition, one of the downstream signaling amplification molecules of NMDAR activation inducing dyskinesia is assumed to be the increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is activated by Ca2+ influx through the NMDAR overactivation and modulated by autophosphorylation at threonine-286 of the CaMKII α isoform (pCaMKII-Thr286) [18], [19]. Whether the antidyskinetic effect of NR2B antagonist CP-101,606 is due to regulating the interactions of NR2B with Fyn, and suppressing NR2B tyrosine phosphorylation and pCaMKII-Thr286 is still unknown. Based on the above observations, the evidences were investigated in the present study.
Section snippets
Subjects
Sixty female Sprague-Dawley rats (weighing 190–260 g) were used in this study. The experiments were in accordance with the ARRIVE guidelines. Rats were kept in a room maintained at a temperature of 22 °C ± 1 °C under a 12-h light/dark cycle and had free access to water and standard rat chow in the Institute of Experimental Animal Science, the Shanghai Jiaotong University School of Medicine. All protocols involving animals were approved by the Institutional Ethics Committee of Shanghai Jiaotong
Effects of CP-101,606 on abnormal involuntary movements (AIMs)
Chronic l-dopa treatment is known to induce orolingual, limb, axial and locomotor AIMs. Our study demonstrated that total AIMs scores presented gradually rise during the period of repeated l-dopa administration in PD rats (LID group), as shown in Fig. 2. On day 23, coadministration of CP-101,606 significantly reduced the total AIMs scores. The total AIMs scores on day 23 averaged 14.2 ± 0.65 in PD rats with repeated l-dopa administration (LID group) in comparison with 9.5 ± 0.42 in rats with
Discussion
Dyskinesia induced by chronic l-dopa treatment has limited l-dopa use for PD treatment. Much research has indicated that chronic l-dopa treatment was liable to result in dyskinesia in the parkinsonian rats. In agreement with one recent report [27], in the current study we found that l-dopa treatment for 22 days lead to all the dyskinesia expression including orolingual, limb, axial and locomotor dyskinesia as presented by increased AIMs. In addition, in our preliminary experiment, we found that
Conclusion
In summary, our data clearly indicated that CP-101,606 which acts to block NR2B may benefit for the therapy of LID through lowering the function of NMDAR via suppressing the interactions of NR2B with Fyn and NR2B tyrosine phosphorylation and further attenuating NMDAR downstream signaling amplification.
Conflict of interests
There are no conflict of interests in the present research.
Acknowledgment
The present research was sponsored by Chinese National Natural Science Foundation (No. 81100954) and Shandong Provincial Natural Science Foundation (ZR2013HQ003).
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