Platelet‐derived growth factor (PDGF)‐BB protects dopaminergic neurons via activation of Akt/ERK/CREB pathways to upregulate tyrosine hydroxylase

Abstract Aims The neurotropic growth factor PDGF‐BB was shown to have vital neurorestorative functions in various animal models of Parkinson's disease (PD). Previous studies indicated that the regenerative property of PDGF‐BB contributes to the increased intensity of tyrosine hydroxylase (TH) fibers in vivo. However, whether PDGF‐BB directly modulates the expression of TH, and the underlying mechanism is still unknown. We will carefully examine this in our current study. Method MPTP‐lesion mice received PDGF‐BB treatment via intracerebroventricular (i.c.v) administration, and the expression of TH in different brain regions was assessed by RT‐PCR, Western blot, and immunohistochemistry staining. The molecular mechanisms of PDGF‐BB‐mediated TH upregulation were examined by RT‐PCR, Western blot, ChIP assay, luciferase reporter assay, and immunocytochemistry. Results We validated a reversal expression of TH in MPTP‐lesion mice upon i.c.v administration of PDGF‐BB for seven days. Similar effects of PDGF‐BB‐mediated TH upregulation were also observed in MPP+‐treated primary neuronal culture and dopaminergic neuronal cell line SH‐SY5Y cells. We next demonstrated that PDGF‐BB rapidly activated the pro‐survival PI3K/Akt and MAPK/ERK signaling pathways, as well as the downstream CREB in SH‐SY5Y cells. We further confirmed the significant induction of p‐CREB in PDGF‐BB‐treated animals in vivo. Using a genetic approach, we demonstrated that the transcription factor CREB is critical for PDGF‐BB‐mediated TH expression. The activation and nucleus translocation of CREB were promoted in PDGF‐BB‐treated SH‐SY5Y cells, and the enrichment of CREB on the promoter region of TH gene was also increased upon PDGF‐BB treatment. Conclusion Our data demonstrated that PDGF‐BB directly regulated the expression of TH via activating the downstream Akt/ERK/CREB signaling pathways. Our finding will further support the therapeutic potential of PDGF‐BB in PD, and provide the possibility that targeting PDGF signaling can be harnessed as an adjunctive therapy in PD in the future.


| INTRODUC TI ON
Tyrosine hydroxylase (TH) is a rate-limiting enzyme in dopamine biosynthesis, which catalyzes the conversion of L-tyrosine to L-DOPA, which is a precursor for dopamine. As a marker for dopaminergic neurons in the central nervous system (CNS), TH are predominately expressed in those cells and are responsible for maintaining the level of DA in the brain. 7,8 Although studies have revealed the regulatory role of TH on DA production, so far, there is no effective medicine to target dopaminergic neurons and improve the expression of TH. As an important endogenous growth factor, the homodimer of the platelet-derived growth factor isoform B (PDGF-BB) has been shown to have restorative effects in the dopaminergic system in vivo. In several classic toxin-induced PD models, it has been shown that intracerebroventricular (i.c.v) application of PDGF-BB for two weeks not only restored DA neurotransmission, but also provided functional recovery of those animals. [9][10][11] Importantly, an increased number of nigral TH + cells, as well as the density of the TH + fiber, along with the partial restoration of DA transporter levels, were also detected in PDGF-BB-treated animal models. 4,10 In vitro studies also indicated that the specific effects of PDGF-BB-mediated TH expression in dopaminergic neurons are promising. 12 However, the molecular mechanism leading to this compelling effect is still unknown. Recently, we have demonstrated that PDGF-BB protects SH-SY5Y cells from neurotoxin MPP + -induced ROS generation and cellular loss. 13 Implicating activation of those pro-survival signaling pathways could be beneficial to the normal function of dopaminergic neurons, such as TH expression.
In this study, we evaluated the effect of PDGF-BB on TH expression in vivo by using the MPTP-lesioned mice model. We also tested the effect of PDGF-BB on TH expression in both primary dopaminergic neuron and dopaminergic neuronal cell line SH-SY5Y cells. Here, we provide evidence to show that the signaling pathways PI3K/Akt, as well as MAPK/ERK, are activated and play an important role in this process. We have also demonstrated that PDGF-BB promotes the expression of TH via triggering the nucleus translocation of CREB, which acts as a transcriptional activator of TH genes in dopaminergic neuronal cells. Our data further evaluated the prominent therapeutic potential of PDGF-BB and the molecular mechanism of PDGF signaling in the dopaminergic system.

| PD animal model and intracerebroventricular (i.c.v.) administration
We used 18 eight-week-old C57BL/6J mice for three groups in this study, and each group contains six animals. To generate the PD mouse model, mice were injected with MPTP intraperitoneally at the dose of 30 mg/kg/day for five days. Since a PD model would exhibit a reduced motor function, the rotarod test was used to evaluate the successful establishment of a PD model five days after the last injection. For PDGF-BB administration, we performed intraventricular infusion via an intracerebroventricular catheter at a dose of 50 ng/ day for seven days. Three weeks later, the mice were sacrificed, and the brain tissues were dissected, fixed, and collected.

| Western blotting (WB)
SH-SY5Y cells or brain tissues were lysed in the RIPA buffer (Solarbio, Cat#R0010) and supplemented with protease inhibitor and phosphatase inhibitor (Roche, Cat#04693132001; 04906837001). The concentration of protein was quantified by a BCA kit (Solarbio, Cat#CA1210). A loading buffer was added in protein samples and boiled at 98°C for 5 min. Equal amounts of protein samples were electrophoresed in a sodium dodecyl sulfatepolyacrylamide gel (10%-12.5%), followed by transferring to PVDF membranes (Millipore). PVDF membranes were blocked with 5% non-fat milk (BD Biosciences, Cat#232100) or 5% bovine serum albumin (Amresco, Cat#0332) for 1 h at room temperature, and the blots were probed with primary antibodies and secondary antibodies. The blots were detected by an HRP chemiluminescence kit (Millipore, Cat#WBKLS0100) under chemiluminescence imaging analysis system (Tanon, 5200). Quantification of the blots was assessed by Image J.

| Immunostaining
Primary neurons were plated on cover slips in a 24-well plate. After the treatment, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Cells were then

| Chromatin immunoprecipitation (ChIP) assay
Cells were cultured in 10 cm dishes and treated (or untreated) with PDGF-BB (50 ng/ml) for 2 h at a density of 80%-90%. Untreated and treated cells were then operated according to the manufacturer's instructions of SimpleChIP ® Enzymatic Chromatin IP Kit (Agarose Bead, CST, 9002). Genome DNA was acquired after cross-link, nuclear processing, and fragmentation. Fragmented DNA was immunoprecipitated by incubation with anti-p-CREB antibody overnight at 4°C. Finally, p-CREB enrichment efficiency was analyzed by PCR assay. Primers targeted to TH gene promoter (forward: 5′-GCTGGGGAGTGAAGGCAAT-3′, reverse: 5′-CATGGCTCAGTGTGGAGGTC-3′) were used for PCR.

| Dual-luciferase reporter assay
The oligonucleotide fragment of TH promoter region was amplified from the genomic DNA of SH-SY5Y cells and inserted into the pGL3-basic luciferase plasmid (Promega) by using In-Fusion cloning technology (Takara). SH-SY5Y cells were transfected with TH promoter region-inserted pGL3-basic luciferase plasmid expressed firefly luciferase enzyme, as well as a pRL-SV40 plasmidencoded renilla luciferase enzyme by using Lipofectamine™ 2000 Transfection Reagent (Invitrogen). Forty-eight hours later, cells were treated (or untreated) with PDGF-BB (50 ng/ml) for 2 h.
The activity of luciferase enzymes was analyzed by using a dualluciferin report detection kit according to the manufacturer's instructions (TransDetect ® , FR201-01). The transcriptional activities were acquired by calculating the ratio of firefly luciferase activity vs. renilla luciferase activity.

| Statistical analysis
All the data are expressed as mean of ±SD. Statistical significance was evaluated by unpaired student's t-test for two groups or oneway ANOVA followed by Bonferroni for multiple comparisons. All experiments were repeated at least three times, and p < 0.05 was considered statistically significant. The experimental data were statistically analyzed and graphed by using GraphPad Software.
Before statistical analysis, the data were tested for Gaussian distribution assessed using D'Agostino-Pearson test. Furthermore, the data failed to show Gaussian distribution has been assessed by nonparametric tests. p values <0.05 were considered statistically significant.

| PDGF-BB increases the expression of TH in MPTP-lesioned mice model in vivo
In our study, we decided to first investigate the effect of PDGF-BB in restoring the expression of TH in the MPTP-lesioned mice model.

| PDGF-BB induces the expression of TH in dopaminergic neuronal cells in the presence of neurotoxin MPP +
From the in vivo study, we confirmed that i.c.v administration of PDGF-BB promoted the expression of TH in the MPTP-lesion PD mice model. Next, we wanted to explore the regulatory effect of PDGF-BB on TH expression in vitro. To do this, we cultured mouse primary dopaminergic neurons and treated cells with PDGF-BB in a time-dependent manner using a dose of 50 ng/ ml, which was shown to effectively protect neuronal cells against MPP + toxicity in our previous study. 13 To examine the effect of PDGF-BB on the expression of TH at both mRNA and protein levels, cells were harvested at indicated time points, respectively. As shown in Figure 2A, the increased transcription of TH gene was observed as early as 30 min after PDGF-BB exposure, which was evidenced by RT-PCR. We also detected that PDGF-BB significantly promoted the expression of TH in primary dopaminergic neurons as shown in Figure 2B, and this effect was confirmed by immunocytochemistry. As shown in Figure 2C Figure 2E). Following this, we wanted to detect the restorative function of PDGF-BB on TH expression in neurotoxin MPP + exposed SH-SY5Y cells, which is a widely used cellular model to study the molecular events related to dopaminergic neuronal loss observed in PD. We pretreated SH-SY5Y cells  Figure 2F, exposure of MPP + significantly reduced the expression of TH in SH-SY5Y cells, while PDGF-BB treatment was able to reverse MPP + caused reduction in TH. Thus, we further confirmed that PDGF-BB is able to upregulate TH expression in MPP + injured dopaminergic neuronal cells.

| Activation of ERK/Akt signal pathways are important for PDGF-BB-mediated TH expression
Our next step was to explore the underlying molecular mechanism of PDGF-BB-mediated upregulation of TH expression in SH-SY5Y cells. Based on existing literature and our previous studies, it has been shown that PDGF-BB mainly activates two intracellular signaling pathways including PI3K/Akt and mitogen-activated protein  Figure   S1). 13 Therefore, we first validated the activation PI3K/Akt and ERK pathways upon PDGF-BB treatment in SH-SY5Y cells. As shown in Figure 3A, we detected a time-dependent increase in phosphorylated Akt after PDGF-BB stimulation in SH-SY5Y cells. We also demonstrated the prominent activation of MAPK/ERK signaling upon PDGF-BB treatment ( Figure 3B), which was evidenced by increased phosphorylation of ERK in SH-SY5Y cells compared with the level in untreated cells. Since PDGF-R are the known surface receptors for PDGF-BB on target cells, by pretreated cells with Imatinib (STI-571), a pharmacological inhibitor of tyrosine kinase including PDGF receptor, PDGF-BB-mediated activation of ERK and Akt signaling were significantly blocked as shown in Figure 3C,D. To further confirm that the effect of PDGF-BB on PI3K/Akt and ERK activation is related to increased expression of TH, the LY294002 (PI3K pathway-specific inhibitor) and U0126 (ERK pathway-specific inhibitor) were used in MPP + -exposed SH-SY5Y cells. According to our results, it was very obvious that the PDGF-BB-mediated reversal effect of TH expression in MPP + treated SH-SY5Y cells was significantly inhibited upon pretreating cells with either the PDGF-R inhibitor STI-571 or the PI3k/Akt and ERK pathways inhibitors, respectively ( Figure 3E).
Thus, this data indicated that activation of the PI3K/Akt and MAPK/ ERK pathways through its cognitive receptor PDGF-R in SH-SY5Y cells is essential for PDGF-BB-mediated upregulation of TH.

| PDGF-BB mediates activation of downstream transcription factor CREB in dopaminergic neuronal cells
To further explore the downstream transcription factors that are responsible for PDGF-BB-mediated upregulation of TH, we analyzed the promoter region of the TH gene and found the presence and assessed for the level of p-CREB, respectively. As shown in Figure 4A,B, PDGF-BB treatment triggered rapid phosphorylation of CREB, which was evidenced by WB. We further examined the location of p-CREB by immunocytochemistry. We also found that upon PDGF-BB stimulation, a significant increase in fluorescent intensity in the nucleus was detected in PDGF-BB-treated SH-SY5Y cells as shown in Figure 4C,D. By performing further studies using the upstream Akt pathway inhibitor LY294002 and ERK inhibitor U0126, we demonstrated that PDGF-BB-mediated activation of CREB in SH-SY5Y cells through activating PI3K/Akt and ERK signaling pathways. As shown in Figure 4E, pretreated cells with these two inhibitors significantly ameliorate PDGF-BB-mediated upregulation of p-CREB in the nucleus in SH-SY5Y cells.

| PDGF-BB induces the recruitment of CREB to the promoters of TH gene in dopaminergic neuronal cells
The cAMP response element (CRE) is a 30 bp DNA fragment with an 8-base palindrome structure of TGACGTCA, which mainly presents in the promoters of target genes ( Figure 5A). Since CREB can bind to the CRE region and regulate TH gene transcription, we then wanted to demonstrate that CREB was directly involved in the transcription of TH genes. For this, we evaluated the recruitment of CREB to the promoter of TH genes by dual-luciferase reporter assays and ChIP assay. For dual-luciferase reporter assays, we inserted the TH promoter fragment amplified from human genomic DNA into the PGL-3 luciferase reporter. Compared with the control group, the luciferase activities were significantly increased in the PDGF-BB-treated group, indicating that PDGF-BB treatment promotes the activated CREB binding to the promoter region of TH genes ( Figure 5B). For the ChIP assay, SH-SY5Y cells were treated with PDGF-BB for 60 min, and after immunoprecipitation of PDGF-BB-treated cellular chromatin fragments using antibodies against CREB. As evident from the PCR and RT-PCR results in Figure 5C Figure 5E). Our results showed that knockdown of CREB by using si-CREB resulted in significant abolishment of PDGF-BBmediated TH induction as shown in Figure 5F. In addition, we also evaluated the functional relevance of CREB in PDGF-BB-mediated TH expression in SH-SY5Y cells in the presence of MPP + . As shown in Figure 5G, PDGF-BB failure to reverse the reduced expression of TH in the CREB knockdown group, indicated that CREB is essential to induce the expression of TH in MPP + impaired dopaminergic neuronal cells. Taken together, these data suggest that a CREB pathway was involved in PDGF-BB-mediated TH expression in dopaminergic neuronal cells, and that this pathway also contributed to the PDGF-BB-mediated protection of dopaminergic neuronal cells from MPP +mediated impairment of TH expression.

| CREB was activated in PDGF-BB administrated MPTP-lesioned mice model
Since we detected the obvious activation of CREB in our in vitro experiments, we wanted to further validate the activation of CREB in the animal model. To do this, we further examined the expression of p-CREB and its co-localization with TH-positive cells in the SNpc region. As shown in Figure 6A-C, we detected a relatively low signal of p-CREB in the SN region of the brain sections from saline-treated mice, while in the MPTP-lesioned group, the number of immunoreactive cells for p-CREB was significantly reduced. However, we detected very high levels of p-CREB in the SNpc region of PDGF-BB administrated MPTP-lesioned mice. Since the administration of PDGF-BB is easy to diffuse to the midbrain, we also collected lower magnification image in that region (Figures S2 and S3). The results between the three groups in the midbrain are similar to that in the SN, which further validated the function of PDGF-BB on CREB activity in vivo. According to the co-localization analysis, our results indicated that MPTP administration led to a dramatic degeneration of the dopaminergic neurons in the SNpc, as well as the impaired CREB activation. However, PDGF-BB administration was able to activate CREB in TH-positive cells and increases the TH-immunoreactive intensity in the MPTP-lesioned group. Thus, we further confirmed the activation of CREB in PDGF-BB administrated MPTP-lesioned mice.  16 Previous studies have indicated that PDGF-BB play a crucial role in the regulation of neurogenesis in both the developing and adult brains. 17 The restorative function of PDGF-BB observed in several PD models was also partially attributed to the improved endogenous neurogenesis elevated upon PDGF-BB administration. 18,19 However, besides the great potential is also a remarkable phenomenon in MPP+ exposed DA neurons. [21][22][23] In our current study, we also used the 1-methyl-4-phenylpyridinium Similarly, we also detected that PDGF-BB treatment was able to reverse MPP+-induced suppression of TH. These data further support the therapeutic potential of PDGF-BB in treating patients with early diagnosis of PD.

| DISCUSS ION
Through binding to its cognate receptor PDGF-Rα or PDGF-Rβ, PDGF-BB has been shown to activate various signaling pathways, such as PI3K/Akt, phospholipase C (PLC) -γ1, and ERK MAPK, in various cell lines and tissues. [24][25][26] Akt is a downstream target of PI3K, and both Akt and ERK signaling molecules play essential roles in multiple cellular processes, such as cell proliferation and migration. PDGF-BB was shown to promote the proliferation and migration of vascular smooth muscle cell through the activation of PDGFRβ/Akt/ERK pathways in several studies. 27,28 Our previous reports have also demonstrated that PDGF-BB ameliorated neurotoxin-mediated impairment of neural progenitor cells (NPCs) proliferation through activation of the ERK MAPK pathway. 29 In this study, we also detected rapid activation of PI3K/Akt and ERK upon PDGF-BB treatment in SH-SY5Y cell. Inhibition of these pathways at a dose that did not affect the basal expression of TH significantly The transcription factor CREB has emerged as a major regulatory factor for neuronal survival mediated by growth factors such as PDGF-BB. 30,31 Our findings also unraveled the key role of CREB in PDGF-BB-mediated induction of TH expression in the presence F I G U R E 6 PDGF-BB exposure results in increased activation of CREB within dopaminergic neurons. (A) Representative image of immunofluorescence staining demonstrating increased p-CREB in dopaminergic neurons within the SNpc region in PDGF-BB administrated mice brain. (B) Quantification of p-CREB and/or (C) TH-positive cells in the substantia nigra from sections collected from saline, MPTPinjured, and PDGF-BB administrated MPTP-injured groups. All data are presented as mean ± SD of three replicates of indicated treatment conditions *p < 0.05 vs. control group, #p < 0.05, ##p < 0.01 vs. MPTP group of MPP+. We detected a significant increase in CREB translocation into the nucleus in SH-SY5Y cells exposed to PDGF-BB compared with the cells not treated with the growth factor. Further dissection of CREB pathway was done by using the pharmacologic inhibitors, wherein it was identified that the PI3K/Akt and ERK pathway were upstream of CREB and played a critical role in PDGF-BB-mediated modulation of TH expression. We also demonstrated that CREB was directly involved in the transcription of TH genes in PDGF-BB-treated neuronal cells by ChIP assay. Also, PDGF-BB has been shown to activate several transcription factors, such as Egr-1 and AP-1 to modulate its function. 32,33 According to relative bioinformatics analysis, the presence of the consensus CRE at the promoter region of the TH gene makes CREB has the greatest potential to be employed for the transcription of TH upon PDGF-BB exposure.
In addition to the upregulation of TH, CREB activation has been involved in other beneficial functions upon PDGF-BB treatment.
Our previous studies demonstrated that CREB is the key regulator for PDGF-BB-mediated NPC proliferation. 29,34 Our earlier studies also demonstrated that activation of CREB is involved in neuroprotection against neurotoxin MPP + induced oxidative stresses. These findings point out that CREB activation might be the key molecular event elicited upon PDGF-BB application and thus can be considered as an alternative therapeutic target in PD treatment.
In summary, we validated the expressional upregulation of TH in MPTP-lesion mice upon i.c.v administration of PDGF-BB for 2 weeks.
The in vitro experiments further dissected the involved molecular mechanism in this process. We demonstrated that PDGF-BB rapidly activated the pro-survival PI3K/Akt and MERK/ERK signaling pathways, and we also provided the first evidence that PDGF-BB promoted TH production via activation of the transcription factor CREB, which translocated to the nucleus and bound to the promoter region of TH genes in dopaminergic neuronal cells. Our findings in this study will further support the possibility that targeting PDGF signaling can be harnessed as an adjunctive therapy in PD in the future.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
LY and WZY contributed to research concept, research administration, and support. CH and TY carried out the experiments with the help of LZH, CXM, GF, and LYZ. CH and TY performed statistical analyses. LY and CH analyzed the data and wrote the manuscript.
All authors edited and approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.