Antidyskinetic Treatment with the mGluR5 Antagonist MPEP Affects CaMKII/CREB/BDNF Molecular Pathways in the Parkinsonian Striatum CURRENT STATUS: POSTED

L-DOPA is still the gold-standard drug for the treatment of Parkinson's disease (PD). However, the long-term therapy often causes L-DOPA-induced dyskinesia (LID). Metabotropic glutamate receptor type 5 (mGluR5) is abundant in the basal ganglia, and its antagonists is thought to alleviates LID, but the underlying mechanisms have remained unclear. We used 6-hydroxydopamine-lesioned rats to create PD rat model, PD rats were daily treated with L-DOPA alone or with MPEP 30 min before L-DOPA for 3 weeks, and at least 21 days of L-DOPA was administrated followed with microinjection of saline, CaMKII antagonist KN-93, anti-CREB, or anti-BDNF into the lesioned striatum of all the PD rats. The behavioral evaluation of abnormal involuntary movements(AIM) and rotational behavior tests were performed on the 2, 9, 11, 18, 21 and 23 days after drug application, and to tested the protein level of mGluR5, CaMKII, CREB and BDNF by western blot. reversed the of and of mGluR5 and in the found that the CaMKII inhibitor KN93, anti-CREB and anti-BDNF intrastriatal injection partly attenuates LID; KN-93 downregulated the striatal p-CaMKII, p-CREB and BDNF expression of the lesioned side, but the striatal mGluR5 expression without inhibition; anti-CREB downregulated the striatal p-CREB and BDNF expression of the lesioned side, but the striatal mGluR5 and p-CaMKII expression without inhibition; anti-BDNF downregulated the striatal BDNF expression of the lesioned side, but the mGluR5, CaMKII and CREB protein PD rat model and MPEP strengthened L-DOPA anti-parkinsonian effects. In this present study, we explored the possible molecular mechanism of the alleviation effect mediated by the antagonist of mGluR5. Our results showed that motor complications induced by administration of L-DOPA are companied by the signaling proteins in 6-OHDA-lesioned hemiparkinsonian rats, while MPEP cotreatment reduced overexpression of striatal mGluR5 and CaMKII/CREB/BDNF in the LID rats. In addition, our results further demonstrated that the striatal mGluR5 expression of the L-DOPA, KN-93, anti-CREB and anti-BDNF groups were remarkably upregulated as compared to control group. KN-93, anti-CREB and anti-BDNF intrastriatal injection partly attenuates LID. CaMKII inhibitor KN-93 downregulated the striatal p-11 BDNF anti-CREB downregulated the striatal p-CREB and BDNF protein expression of the lesioned side, but the striatal mGluR5 and p-CaMKII protein expression without inhibition. anti-BDNF downregulated the striatal BDNF expression of the lesioned side, but the mGluR5, CaMKII and CREB expression without inhibition. Taken together, our results provide evidence that mGluR5 specific antagonist MPEP attenuates L-DOPA-induced dyskinesia through affects CaMKII/CREB/BDNF molecular pathways in this experimental model of LID. in MPTP-lesioned The underlying molecular mechanisms of the anti-dyskinetic activity of mGluR5 antagonists remain unknown. Our results showed that cotreatment of MPEP with L-DOPA to PD rats not we examined the effects of specific CaMKII inhibitor KN-93, anti-CREB, and anti-BDNF on LID and the markers of the mGluR5-related CaMKII/CREB/BDNF signaling pathway in the PD model rats. Our data showed that KN-93, anti-CREB and anti-BDNF intrastriatal injection partly attenuates LID, and accordingly downregulated the striatal p-CaMKII, p-CREB and BDNF protein expression of the lesioned side, while mGluR5 protein expression without inhibition. Taken together, these findings suggested that the mGluR5 specific antagonist MPEP attenuates L-DOPA-induced dyskinesia through affects CaMKII/CREB/BDNF molecular pathways in this experimental model of LID. However, further studies, using striatal-specific CaMKII/CREB/BDNF KO or viral-delivered RNAi against CaMKII/CREB/BDNF in this region, whether the increase in CaMKII/CREB/BDNF expression In summary, this study suggested that the selective mGluR5 antagonist MPEP alleviated the abnormal involuntary movements, reversed the reduction of rotational response duration, and inhibited the overexpression of striatal mGluR5 and CaMKII/CREB/BDNF induced by L-DOPA treatment rats in the lesioned striatum. Furthermore, these results provide evidence that KN-93, anti-CREB and anti-BDNF partly attenuates AIM, accordingly inhibited the hyperactivity of striatal CaMKII/CREB/BDNF protein expression in the lesioned striatum of LID rats. Therefore, these results suggested that mGluR5 specific antagonist MPEP attenuates L-DOPA-induced dyskinesia through affects CaMKII-CREB-BDNF


Introduction
Parkinson's disease (PD), one of most common neurodegenerative diseases, the loss of dopaminergic neurons in the substantia nigra pars compacta are main neuropathological features, resulting in significantly decreased dopamine levels in the striatum (Bastide et al., 2015). Although L-DOPA is still the gold-standard drug of dopamine replacement treatment for PD, however, the long-term L-DOPA therapy causes a decrease in the efficacy and disabling abnormal involuntary movement (AIM), namely L-DOPA-induced dyskinesias (LID), which greatly hampered the use of L-DOPA in PD treatment (Fabbrini et al., 2007). At present, the underlying molecular mechanisms of LID are still elusive. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is ubiquitously expressed in the central nervous system and is particularly abundant at excitatory synapses. This kinase plays an important role in the regulation of neuronal survival and death (Zhang et al., 2017b). CaMKII and its interacting partners are also believed to play a critical role in the pathogenesis of various neurological and neurodegenerative disorders, such as PD. Recent research show that chronic L-DOPA treatment activates CaMKII in striatal neurons (Gan et al, 2015), and the pharmacological inhibition of the CaMKII with a selective inhibitor KN93 could ameliorated dyskinesia in a rat PD model (Zhang et al., 2014;Yang et al, 2018). However, the specific molecular mechanisms of the involvement of CaMKII in LID are not fully unclear. cAMP-response element binding protein (CREB) is another intracellular signaling that its activation prevents dopaminergic cell death in PD (Ham et al., 2017). Innovative treatment strategies for PD, especially at its early stages, have gained paramount clinical attention (Beitz, et al., 2014). Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family of proteins, is a major promoter of synaptic plasticity, and regulates neuronal proliferation, differentiation and survival (Binder and Scharfman, 2004;Cohen-Cory et al., 2010;Park and Poo, 2013). The recent research indicates that L-DOPA administration induces BDNF release from corticostriatal fibers, which in-turn increases the expression of D3 receptors, and the D3 expression increase plays a key role in the emergence of LID (Guillin et al., 2001). Furthermore, recent findings demonstrate that repeated L-DOPA therapy enhanced the levels of c-fos and BDNF mRNA in the dopamine-depleted subthalamic nucleus in the PD model rat (Zhang et al., 2006).
It is of great importance to investigate what the molecular alterations in the mGluR5-related signaling pathway are, in order to interpret the antidyskinesia effect of the antagonists of mGluR5. This study aimed to investigate the effects of mGluR5 specific antagonist MPEP on LID, and to tested the protein level of mGluR5, CaMKII, CREB and BDNF in the mGluR5 mediated CaMKII/CREB/BDNF signaling pathway.

Materials And Methods Animals
Male adult Sprague-Dawley animals (weighing 180-220 g) were obtained from the Center for Experimental Animals, Soochow University,China. The maintenance of the animals followed the guidelines of the National Institutes of Health for the care and use of laboratory animals. All experimental protocols involving animals were approved by the ethics committee of the Second Affiliated Hospital of Soochow University. The dose of L-DOPA was selected according to our previous study (Huang et al., 2011), while the dose for MPEP was used based on another study (Levandis et al., 2008).
Moreover, Fifty successfully PD model rats were randomly classified into five groups (n = 10 for each group): (1) Intraperitoneal (i.p.) injection with saline (the control group) for 23 days (n = 10). The other forty rats were treated with L-DOPA (25 mg/kg L-DOPA plus 6.25 mg/kg benserazide in saline) to 21 days, at the 22 day, L-DOPA-treated and dyskinetic rats were randomly divided into four groups (n = 10 for each group), these rats received intrastriatal administration of (2)  indicates existence during less than half one minute; 2 indicates existence during over half one minute; 3 indicates persistent existence but inhibited by external stimuli; 4 indicates persistent existence without the inhibitory possibility. during 140 minutes after L-DOPA administration, rats were assessed every 20 minutes individually. Each rat was rated for appearance of abnormal movements in axial, limb, orolingual movements. Each AIM was quantified within one minute and present as time which the rat stayed for each movement. The total scores of axial, limb and orolingual AIM were regarded as ALO AIM scores.
Rotational behavior was measured immediately following the treatment of L-DOPA. The duration of the rotational response was considered the time between the first 5 min interval when turning exceeded 20% of the peak rate and the first interval when turning fell below 20% of the peak rate.
The peak intensity of rotation was measured as the peak number of contralateral turns in any 5 min interval.
The behavioral evaluation of AIM, and rotational behavior tests were performed on the 2, 9, 11, 18 and 21 days after L-DOPA application. The AIM evaluation was performed on the 21 and 23 days after drug administration.

Western Blotting
Rats were anesthetized with 10% chloral hydrate (0.5 ml/100 g) for 30 min after the last administration, rats were sacrificed and their brains were quickly taken out. Then the lesioned striatum were separated and stored at -70 °C for Western blots. The tissues from the dorsal striatum were homogenized and centrifuged at 12000 g for 30 min. The protein concentration was determined by a BCA assay kit (Pierce Chemical, Rockford, IL). A total of 60 µg of protein lysates from each sample were added into the loading buffer, mixed and heated at 95 °C for 5 min. Then the samples were loaded into SDS polyacrylamide electrophoresis gels (8%) and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The resultant blots were blocked in Tris buffered saline/Tween 20 (TBST) buffer (10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween-20, pH 8.0) with 5% milk for 1 h. The membranes were then incubated with the indicated primary antibodies (rabbit anti-mGluR5, 1:2000, or rabbit anti-P-CaMKII/CaMKII, 1:1000, or rabbit anti-P-CREB/CREB, 1:1000, or rabbit anti-BDNF, 1:2000, or mouse anti-β-actin, 1:5000) at 4 °C overnight. Next, the membranes were rinsed with TBS-T for three times and incubated with appropriate secondary antibodies for 1 h at room temperature.
At last, the membrane was briefly washed and subjected to ECL chemiluminescence exposure (GE healthcare, Buckinghamshire, UK). Images were obtained on a computer-aided CCD camera. Optical density (OD) was detected with Mercator software (Explora Nova). All individual protein bands were compared with their internal control actin values in order to provide relative protein abundance. All the procedures were repeated 3 times.

Statistical analysis
All data were expressed as mean ± standard error of the mean (SEM). Behavioral assessments and biochemical data analysis among different groups were performed using one-way analysis of variance (ANOVA) coupled with the LSD's post hoc analysis (SPSS, 18.0, Chicago, IL). P < 0.05 was viewed as

MPEP attenuates abnormal involuntary movements induced by L-DOPA
To mimic dyskinesia phenomena in PD patients, we consecutively treated PD model rats with L-DOPA for 21 days, and observed progressive AIM. Whereafter, we examined the potential role of mGluR5 in AIM. By co-treatment of L-DOPA with the specific mGluR5 antagonist MPEP, we found the alleviation of these behavioral abnormalities. Specifically, at 21 day after L-DOPA administration, the axial AIM score of MPEP plus L-DOPA group was significantly decreased than that of L-DOPA treatment group (P < 0.05, Fig. 1a). Similarly, the limb AIM score of the coadministration group was markedly reduced at 18 and 21 day as compared to the L-DOPA treatment group (P < 0.01, Fig. 1b). Moreover, the orolingual AIM score of the coadministration group was significantly decreased at 21 day as compared to the L-DOPA treatment group (P < 0.01, Fig. 1c). Interestingly, the total AIM score of the coadministration group were reduced at 18 and 21 day as compared to the L-DOPA treatment group (P < 0.05, Fig. 1d). Lesioned rats with received of saline (controls, n = 10) or MPEP (n = 10) alone failed to develop AIM. Take together, our results indicated that inhibition of mGluR5 by MPEP prevents L-DOPA-induced AIM in 6-OHDA-lesioned rats.
MPEP ameliorated the reduction of rotational response duration induced by L-DOPA To mimic the "wearing-off" phenomena in PD patients, we consecutively treated PD model rats with L-DOPA for 21 days, and observed progressive shortening in the duration of rotational response. Our results revealed that administration of L-DOPA alone, given twice daily for 21 days, significantly reduced the rats' rotational response duration; the duration of the rotational response reduced from 138.5 ± 3.6 min to 101.5 ± 5.1 min during the 21-day treatment period (F = 7.991, F = 13.833, F = 35.661, P < 0.05) (Fig. 2). By contrast, coapplication of MPEP with L-DOPA maintained the rotational response duration throughout the 21-day treatment period (P > 0.05) (Fig. 2). This effect was maintained until the end of MPEP with L-DOPA administration and the rotational duration of four time points were not different during the course of the treatment, as compared to L-DOPA treatment group at day 2 (P > 0.05) (Fig. 2). Lesioned rats that injection saline (controls, n = 10) or MPEP (n = 10) alone did not develop rotational response at all.

KN-93, anti-CREB, anti-BDNF attenuates L-DOPA-induced dyskinesia and affects striatal
CaMKII/CREB/BDNF expression Our behavioral results and western blot analysis suggested LID may be due to mGluR5-related higher CaMKII/CREB/BDNF expression. However, how these abnormal molecular pathways alterations influence the contribution of mGluR5 to LID needs to be further investigated. We consecutively treated PD model rats with L-DOPA for 21 days, and observed progressive AIM. By treatment of the specific CaMKII inhibitor KN-93, anti-CREB, anti-BDNF at the 22 day, we found the alleviation of these behavioral abnormalities. Specifically, at 23 day after L-DOPA application, the axial AIM score of anti-CREB and anti-BDNF groups were reduced than that of 21 day (P < 0.05, Fig. 4a). Similarly, the limb AIM score of KN-93 and anti-BDNF groups were significantly reduced than that of 21 day (P < 0.05, Fig. 4b). Moreover, the orolingual AIM score of the KN-93 and anti-CREB groups were decreased at 23 day as compared to 21 day (P < 0.05, Fig. 4c). Interestingly, the total AIM score of the KN-93 and anti-CREB groups were significantly decreased on the 23 day, as compared to 21 day (P < 0.05, Fig. 4d). Nevertheless, the limb, orolingual and total AIM score of the L-DOPA group was significantly increased at 23 day as compared to 21 day (P < 0.05, Fig. 4). In addition, we further examined that the striatal mGluR5 expression of the L-DOPA, KN-93, anti-CREB and anti-BDNF groups were upregulated as compared to control group (P < 0.05, Fig. 5a,b). CaMKII inhibitor KN-93 downregulated the striatal p-CaMKII, p-CREB and BDNF protein expression of the lesioned side, but the mGluR5 expression without inhibition (P < 0.05, Fig. 5a,c). CREB inhibitor anti-CREB downregulated the striatal p-CREB and BDNF protein expression of the lesioned side, but the striatal mGluR5 and p-CaMKII expression without inhibition (P < 0.05, Fig. 5a,d). BDNF inhibitor anti-BDNF downregulated the striatal BDNF expression of the lesioned side, but the mGluR5, CaMKII and CREB expression without inhibition (P < 0.05, Fig. 5a,e). Collectively, these data suggested that the mGluR5 antagonist MPEP attenuates L-DOPA-induced dyskinesia through affects CaMKII/CREB/BDNF molecular pathways in the Parkinsonian striatum.

Discussion
Glutamate plays a key role in the mammalian brain, serving as most of synaptic transmission in the Our study showed that LID may be due to mGluR5-related higher CaMKII/CREB/BDNF expression.
However, how these abnormal molecular pathways alterations influence the contribution of mGluR5 to LID needs to be further investigated. By using behavioral tests and western blot analysis, we

Consent for publication
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Availability of data and materials
The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Competing interests
The authors declare that they have no competing interests.