Amphiregulin blockade decreases the levodopa‐induced dyskinesia in a 6‐hydroxydopamine Parkinson's disease mouse model

Abstract Background Levodopa (L‐DOPA) is considered the most reliable drug for treating Parkinson's disease (PD) clinical symptoms. Regrettably, long‐term L‐DOPA therapy results in the emergence of drug‐induced abnormal involuntary movements (AIMs) in most PD patients. The mechanisms underlying motor fluctuations and dyskinesia induced by L‐DOPA (LID) are still perplexing. Methods Here, we first performed the analysis on the microarray data set (GSE55096) from the gene expression omnibus (GEO) repository and identified the differentially expressed genes (DEGs) using linear models for microarray analysis (Limma) R packages from the Bioconductor project. 12 genes (Nr4a2, Areg, Tinf2, Ptgs2, Pdlim1, Tes, Irf6, Tgfb1, Serpinb2, Lipg, Creb3l1, Lypd1) were found to be upregulated. Six genes were validated on quantitative polymerase chain reaction and subsequently, Amphiregulin (Areg) was selected (based on log2 fold change) for further experiments to unravel its involvement in LID. Areg LV_shRNA was used to knock down Areg to explore its therapeutic role in the LID model. Results Western blotting and immunofluorescence results show that AREG is significantly expressed in the LID group relative to the control. Dyskinetic movements in LID mice were alleviated by Areg knockdown, and the protein expression of delta FOSB, the commonly attributable protein in LID, was decreased. Moreover, Areg knockdown reduced the protein expression of P‐ERK. In order to ascertain whether the inhibition of the ERK pathway (a common pathway known to mediate levodopa‐induced dyskinesia) could also impede Areg, the animals were injected with an ERK inhibitor (PD98059). Afterward, the AIMs, AREG, and ERK protein expression were measured relative to the control group. A group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group. Conclusion Taken together, our results indicate unequivocal involvement of Areg in levodopa‐induced dyskinesia, thus a target for therapy development.


Methods:
Here, we first performed the analysis on the microarray data set (GSE55096) from the gene expression omnibus (GEO) repository and identified the differentially expressed genes (DEGs) using linear models for microarray analysis (Limma) R packages from the Bioconductor project. 12 genes (Nr4a2, Areg, Tinf2, Ptgs2, Pdlim1, Tes, Irf6, Tgfb1, Serpinb2, Lipg, Creb3l1, Lypd1) were found to be upregulated. Six genes were validated on quantitative polymerase chain reaction and subsequently, Amphiregulin (Areg) was selected (based on log2 fold change) for further experiments to unravel its involvement in LID. Areg LV_shRNA was used to knock down Areg to explore its therapeutic role in the LID model.

Results:
Western blotting and immunofluorescence results show that AREG is significantly expressed in the LID group relative to the control. Dyskinetic movements in LID mice were alleviated by Areg knockdown, and the protein expression of delta FOSB, the commonly attributable protein in LID, was decreased. Moreover, Areg knockdown reduced the protein expression of P-ERK. In order to ascertain whether the inhibition of the ERK pathway (a common pathway known to mediate levodopainduced dyskinesia) could also impede Areg, the animals were injected with an ERK inhibitor (PD98059). Afterward, the AIMs, AREG, and ERK protein expression were measured relative to the control group. A group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group.

| INTRODUC TI ON
Parkinson's disease (often known simply as PD) is one of the most prominent kinds of neurodegenerative disease with unexpected intricacies. The neuropathology of this neurodegenerative disease is very well characterized; however, the etiology of the disease is still unknown, which makes it challenging to target therapy. Levodopa (L-DOPA) is reputed to be the most effective drug for improving movement impairments in Parkinson's disease. By replacing the dopaminergic neurons in the substantia nigra that were lost due to degeneration, this treatment can improve motor symptoms. 1 Unfortunately, after using L-DOPA for a while, most persons with PD develop abnormal involuntary movements (AIMs), often known as L-DOPA-induced dyskinesia (LID). 2,3 Dyskinesia is a side effect of L-DOPA treatment that worsens with continued use, negating the drug's therapeutic effects. The mechanisms underpinning levodopainduced motor swings and dyskinesia remain controversial. 4 One acknowledged assertion for this phenomenon is the engagement of both presynaptic and postsynaptic pathways, which ultimately result in non-physiologic excitation of the pulsatile dopamine receptor, leading to a variety of dysfunctional neuronal behaviors. 5,6 Nonetheless, a luculent molecular encompassment of levodopainduced dyskinesia is perplexing, and indeed studies demonstrate that even its evolution is quite capricious. Understanding the molecular signatures of levodopa-induced dyskinesia could pave the way to anti-dyskinetic novel therapy development to halt the impact of dyskinesia in Parkinson's disease. To unravel this, we used the Linear Models for Microarray Analysis (LIMMA) R tool from the Bioconductor project to analyze the microarray data set (GSE55096) from the gene expression omnibus (GEO) repository and identified the differentially expressed genes (DEGs). Twelve genes were found to be upregulated (Nr4a2, Areg, Tinf2, Ptgs2, Pdlim1, Tes, Irf6, Tgfb1, Serpinb2, Lipg, Creb3l1, and Lypd1). Subsequently, Amphiregulin (Areg) was selected (based on log2 fold change) for further experiments to unravel its involvement in LID. Western blotting and immunofluorescence results show that Areg is significantly expressed in the LID group relative to the control. Dyskinetic movements in LID mice were alleviated by Areg knockdown, and the protein expression of delta FOSB, the commonly attributable protein in LID, was decreased. In order to ascertain whether the inhibition of Extracellularsignal-regulated kinase (ERK) pathway (this pathway is commonly known to mediate the levodopa-induced dyskinesia) could also impede Areg, the animals were injected with ERK inhibitor (PD98059).
Afterward, the AIMs, AREG, and ERK protein expression were measured relative to the normal saline group. A group treated with ERK inhibitor had a significant decrease in AREG and P-ERK protein expression relative to the normal saline group. Taken together, our results indicate an equivocal involvement of Areg in levodopa-induced dyskinesia, thus a target for therapy development.

| Microarray data
The microarray expression profiling dataset GSE55096, deposited by Heiman et al., was retrieved from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/). 7 The Gene Expression Omnibus database at the National Center for Biotechnology Information (NCBI) and Array Express at the European Bioinformatics Institute (EBI) are the two major public databases of microarray data. The GEO database has offered a robust platform for the field of bioinformatics, which has allowed researchers to investigate novel biomarkers for the diagnosis, therapy, and analysis of cancer prognosis. 8 The original data was centered on the GPL1261 Affymetrix Mouse Genome 430 2.0 array platform. The experiment contained 77 samples consisting of 44 subjects of direct spiny projection neurons (dSPNs) (direct pathway) and 33 indirect spiny projection neurons (iSPNs) (indirect pathway). The mouse model used in this data set is validated and it uses the green florescence to tag these pathways (Refer to Supporting Information of the metadata). This was further categorized into 6-hydroxydopamine (6-OHDA) Parkinson's disease model or mock lesion (Ascorbate). The 6-OHDA was further subdivided into chronic high levodopa, chronic low levodopa, or chronic saline.
GEO2R is an online tool for identifying differentially expressed molecules across various experimental conditions. 9 It performs comparisons on original submitter-supplied processed data tables using the GEO query and Limma R packages from the Bioconductor project. 10 Bioconductor is an open-source software project based on the R programming language that provides tools for the analysis of highthroughput genomic data. 11 The data from three groups (Chronic high levodopa, chronic low levodopa, and chronic saline) were first visualized in Uniform Manifold Approximation and Projection plot Conclusion: Taken together, our results indicate unequivocal involvement of Areg in levodopa-induced dyskinesia, thus a target for therapy development.

K E Y W O R D S
Amphiregulin, dyskinesia, levodopa, Parkinson's disease (UMAP). Only the direct pathway yielded clear differences between these three groups on the UMAP plot. Hence, our further analysis and validation were performed on the direct pathway. After differential gene expression analysis, each sample's genes fulfilling the following conditions were retained: (1) Upregulated (p-adjusted value ˂0.05, log2 fold change ≥1.5), (2) Downregulated (p-adjusted value ˂0.05, log2 fold change ≤ −1.5). The volcano plot of each sample against the other was plotted in the GEO2R tool. A web-based tool Venny 2.1 (http://bioin fogp.cnb.csic.es/tools/ venny/ index.html) was used to construct a Venn diagram. Heml tool was used to generate a heatmap of the DEGs.  After the injection, the needle remained in place for 5 min before being retracted.

| Open field test
Open field test was performed as we previously elaborated. 12 In a nutshell, mice were first injected with apomorphine (0.5 mg/kg, intraperitoneally). Subsequently, animals' rotation and distance traveled were digitally recorded and analyzed using the ANY-maze video-tracking system (Muromachi Kikai, Tokyo, Japan).
These lentiviral particles were handed as viral particles that were ready to be used for gene silencing. The particles carried 3 to 5 expression constructs, of which each included a target-specific 19-25 nucleotide sequence with a hairpin to produce a shRNA intended to inhibit gene expression. More information on how the Areg LV_shRNA plasmid was constructed may be found in Additional

| Levodopa injection and AIMS observation
Animals that were successfully modeled for Parkinson's disease were divided into three groups. The first group was administered an intraperitoneal injection of a mixed solution of levodopa (12 mg/kg, Cat.

| RNA extraction and real-time quantitative polymerase chain reaction
Mice were executed by cervical dislocation, and the brains were quickly removed and dropped into ice-cold saline. On an icy plastic surface, the left striatum and midbrain were then dissected.
Quantitative polymerase chain reaction was performed using the previously reported approach. 12 Samples were quickly minced in 1 mL of TRIzol. The samples were then put into a 1.5 mL centrifuge tube, to which 200 μL of chloroform was added, properly mixed, and incubated on ice for 10 min before centrifuging for 15 min at 12,000× g, 4°C. After being centrifuged at 12,000× g for 10 min at 4°C, the supernatant was transferred to a 1.5-mL tube free of RNAse, gently mixed with 0.5 mL of isopropyl alcohol, and incubated at room temperature for 10 min. The RNA precipitate was centrifuged at 7500× g for 5 min at 4°C after being washed with 1 mL of 75% ethanol and the supernatant was discarded. The centrifuge tube was turned upside down, dried on a spotless work surface, and then the RNA was dissolved in 40 μL of DEPC-treated water. A spectrophotometer (Thermo, Boston, MA, USA) was used to measure the optical density of the RNA sample at 260 and 280 nm in order to calculate its concentration. After balancing the mRNA concentration, PrimeScriptTM, RT Master Mix was used to reverse-transcribe the mRNA into cDNA. The temperature settings for the PCR were 25°C for 10 min, 42°C for 30 min, and 85°C for 5 min. The primers used are indicated in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal control, and the results were analyzed with a delta-delta Ct method.

| Western blot
Striatum and ventral midbrain proteins were extracted as described in our earlier work. 12 In essence, animals were sacrificed, their brains were swiftly removed, and the ipsilateral ventral midbrain was dissected, instantly stored on dry ice for immediate use, and then placed in a refrigerator set at −80°C for later use.  Table 2. The membranes were treated with the corresponding secondary antibodies the following day (Table 2).
After blotting, an image analyzer was used to scan and review the filter bands (odyssey scanner-USA). Three western blotting F I G U R E 1 Analysis of the microarray data set (GSE55096) using Linear Models for Microarray Analysis (LIMMA). A clear discrimination appears between the chronic saline and chronic levodopa (Low/High). A further analysis of each group against the other indicates the upregulated genes (red color) and the downregulated genes (blue color). The condition used here was p-adjusted ˂0.05. A total of 1049 genes were found to participate in the three treatment groups. UMAP: Uniform Manifold Approximation and Projection plot, Padj: padjusted value.
repeats were examined at least, and the results were displayed in box plots with data points.

| Immunofluorescence staining
After anesthesia with pentobarbital (45 mg/kg) and heart perfusion (with 100 mL of PBS followed by 150 mL of cold 4% paraformaldehyde), the brains were collected and cryoprotected in a solution of 20% (for 24 h) and 30% (for 24 h) sucrose in 0.1 M phosphate buffer at 4°C. The striatum was cut into coronal sections that were 16 μm thick, and they were left to air dry for an entire day. Following that, the sections were blocked for 2 h with 0.1% Triton X-100 and 10%

| Statistical analysis
The statistical analysis was performed using GraphPad Prism for Windows, Version 9.0.0 (GraphPad Software, San Diego, CA, USA).
Groups were compared using Student's t-test. Results are shown as a mean ± standard deviation (SD). At a significance level of p ˂ 0.05, the results were regarded as significant.

| Analysis of the microarray data set (GSE55096) using LIMMA informs 12 differentially upregulated genes involved in levodopa-induced dyskinesia
In order to ascertain the differentially expressed genes associated with levodopa-induced dyskinesia, analysis of GSE55096 data set was done using the LIMMA package from Bioconductor. The data set included three different treatment groups (Chronic saline, Chronic low levodopa, and chronic high levodopa). The UMAP dimension reduction technique was used to explore the clusters. Analysis of each group against the other to reveal the upregulated and downregulated in the volcano plot is indicated in Figure 1. In total, 1049 significant genes (padj ˂ 0.05) were found in both three groups.
Subsequently, these genes were further analyzed using more crite-

| Amphiregulin is highly expressed in the levodopa-induced dyskinesia 6-OHDA Parkinson's disease mouse model
To further explore the protein expression of some of these differentially expressed genes, we chose to investigate Amphiregulin (Areg) because of its central role in inflammation. 15 Neuroinflammation is thought to be involved in the pathogenesis of levodopa-induced dyskinesia. 16

| AREG+ are highly expressed in D1R but not in D2R
Next, we sought to determine the dopamine receptor family to (Arrows in Figure 7). This suggests that D1R is indispensable for AREG in LID.

| Knocking down Amphiregulin significantly decreases the AIMs owing to chronic levodopa treatment
Next, we sought to explore whether knocking down of Amphiregulin prior to levodopa treatment could decrease the AIMs.

| Inhibition of the ERK pathway reduces the expression of Amphiregulin
ERK pathway has been undoubtedly reported to mediate levodopainduced dyskinesia. However, whether the inhibition of this pathway could also impede Areg which we report here as a crucial gene that facilitates dyskinetic movements secondary to chronic levodopa treatment remains to be elucidated. To this end, dyskinesia was first induced by injecting levodopa for 15 days. On the 15th day, animals were divided into two groups. The first group of dyskinetic animals (control) received normal saline 1 h before the next levodopa injection (Normal saline + LID). The second group of dyskinetic animals received ERK inhibitor (PD98059) 1 h before the next levodopa injection. Subsequently, the AIMs were observed and recorded. This process was repeated for four more days, making a total of 5 days for ERK inhibition and 19 days for levodopa injection. The results indicate that a group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group (p = 0.0003, t = 11.82, df = 4) Figure 9A-C.
On AIMS analysis, the results show that the AIMs decreased significantly in a group injected with ERK inhibitor compared to that of normal saline ( Figure 9D). Figure 9E indicates an extended observation and recording (2 hours) on the third day. In a manner similar to this, when we checked the expression of ERK in animals with Areg knockdown, we found that phosphorylated ERK reduced dramatically compared to the group injected with the scrambled virus ( Figure 9F,G).

| DISCUSS ION
Exploring the possible causes of LID is critical in this dispensation where L-DOPA still serves as the mainstay for Parkinson's disease treatment. The emergence of dyskinesia after several weeks or F I G U R E 5 Differentially expressed genes validated for mice brain samples of levodopa-induced dyskinesia are hyper-expressed in the striatum but not in the midbrain. The striatum and midbrain were extracted and processed for quantitative polymerase chain reaction. The results show that these genes are significantly upregulated in the striatum (A-F) but not in the midbrain of levodopa-induced dyskinetic mice (G-L). The data are depicted as the mean ± SD (n = 15). Student's t-test, **p < 0.01. months of L-DOPA treatment has confounded the understanding of its pathophysiology. There have been ramified attempts to uncover how L-DOPA induces dyskinesia. Here, we started by analyzing the data set that was published on the gene expression omnibus repository. We used the criteria (p-adjusted value ˂0.05, log2 fold change ≥1.5) or (p-adjusted value ˂0.05, log2 fold change ≤−1.5) in establishing the upregulated and downregulated genes, respectively. Microarray data sets have long been valuable in identifying genes involved in cancer 20 and neuroinflammation. 21,22 Our findings revealed 12 differently upregulated genes.
The meta-data 7 used in this study included low-dose (2 mg/kg) and high-dose (6 mg/kg) L-DOPA. The rationale for utilizing two distinct doses (low/high) was to ascertain whether the dyskinetic move- It is worth noting that, of the 12 genes on our list, Nurr1 has recently been linked to LID, and upregulation of Nurr1 in the striatum perpetuates dyskinetic actions in a LID model. 27 Our exhaustive Our results show that Areg, Nr4a2(Nurr1), Tinf2, Ptgs2, Pdlim1, and Tes are hyper-expressed in the striatum but not the midbrain.
According to the existing literature, LID-associated abnormal activity and molecular disruptions have been reported in various brain areas. Early investigations, for example, suggest that the primary motor cortex is involved in LID-related dyskinetic movements. [29][30][31] Furthermore, gene expression changes in the primary somatosensory cortex 32 indicate maladaptive neuroplasticity after long-term levodopa administration. Our findings are in line with previous literature, which shows that FOSB is dramatically expressed in the striatum of a LID model. 33,34 The hypothesis that an increase in dopamine release after L-DOPA injection leads first to unusual changes in the striatum, a brain region that contains dopaminergic fibers from midbrain dopaminergic neurons, could explain why Areg, Nr4a2, Tinf2, Ptgs2, Pdlim1, and Tes are expressed in the striatum but not the midbrain. 35 Notably, Areg, Nr4a2, and Ptgs2 expression tended to increase in a comparable manner in both low and high doses of L-DOPA, whilst Pdlim1 and Tes expression were much lower in lowdose L-DOPA. This could be due to variances in the cells that express these genes and their responses to the dosage administered.
We chose to investigate Amphiregulin's involvement in LID because of the available evidence that Areg plays an important role in inflammation. 15 Neuroinflammation is thought to be involved in the pathogenesis of levodopa-induced dyskinesia. 16,17 Our findings F I G U R E 8 Knocking down Amphiregulin significantly decreases the abnormal involuntary movements owing to chronic levodopa treatment. The results indicate that the Areg knockdown group had a significant decrease in Areg mRNA (A). When the levodopa was injected in order to cause dyskinesia, the Areg knocked down group showed an inhibited Areg protein expression (B, C), and consequently a significant decrease in total abnormal involuntary movements score is relative to the scramble injected group (D, E). On immunofluorescence, AREG-positive neurons decreased considerably in a knockdown group compared to the scramble-injected (F, G). The data are depicted as the mean ± SD (n = 10). Student's t-test, **p < 0.01, ***p < 0.001.
show that Areg is pervasive in the levodopa-induced dyskinesia of a  39 Similarly, AREG was discovered to activate the ERK MAPK in dental pulp stem cells, which in turn promoted their differentiation into odontoblasts. 40

F I G U R E 9
Inhibition of the Extracellular-signal-regulated kinase (ERK) pathway reduces the expression of Amphiregulin. A group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group (A-C). On AIMS analysis, the results show that the abnormal involuntary movements decreased significantly in a group injected with ERK inhibitor compared to that of normal saline (D). (E) indicates an extended observation and recording (2 hours) on the third day. In a manner similar to this, when we checked the expression of ERK in animals with Areg knockdown, we found that phosphorylated ERK reduced dramatically compared to the group injected with the scrambled virus (F, G). The data are depicted as the mean ± SD (n = 10). Student's t-test, **p < 0.01.
ERK-MAPK pathway has been undoubtedly reported to mediate the levodopa-induced dyskinesia. Forced activation of dopamine receptor 1 (D1R), as reported by Picconi et al., 41 leads to the phosphorylation of ERK, a key component of LID. 41 In a similar study, both ERK phosphorylation and FosB expression increased after prolonged L-DOPA treatment. 42 It appears that the increases in ERK activation and FosB expression are specific to the exquisitely sensitive reactivity to L-DOPA induced by the dopaminergic lesion, as neither acute nor chronic L-DOPA administration boost ERK phosphorylation in the dopamine-intact striatum. 42 The current findings show that inhibiting this route reduces Amphiregulin expression, which we establish as a key player gene in LID.
In conclusion, our findings suggest that Areg plays a conspicuous role in levodopa-induced dyskinesia, making it a potential therapeutic target.

AUTH O R CO NTR I B UTI O N S
In

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors claim they have no conflicting interests.