Transplantation of Nurr1‐overexpressing neural stem cells and microglia for treating parkinsonian rats

Abstract Background Neural stem cells (NSCs) transplantation is considered a promising treatment for Parkinson's disease. But most NSCs are differentiated into glial cells rather than neurons, and only a few of them survive after transplantation due to the inflammatory environment. Methods In this study, neural stem cells (NSCs) and microglial cells both forced with the Nurr1 gene were transplanted into the striatum of the rat model of PD. The results were evaluated through reverse transcription polymerase chain reaction (RT‐PCR), Western blot, and immunofluorescence analysis. Results The behavioral abnormalities of PD rats were improved by combined transplantation of NSCs and microglia, both forced with Nurr1. The number of tyrosine hydroxylase+ cells in the striatum of PD rats increased, and the number of Iba1+ cells decreased compared with the other groups. Moreover, the dopamine neurons differentiated from grafted NSCs could still be detected in the striatum of PD rats after 5 months. Conclusions The results suggested that transplantation of Nurr1‐overexpressing NSCs and microglia could improve the inhospitable host brain environments, which will be a new potential strategy for the cell replacement therapy in PD.


| INTRODUC TI ON
Parkinson's disease (PD) is a common neurodegenerative disease prevalent in older persons. It is characterized by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta and the striatal DA deficiency. 1 Current therapies for PD are mainly aimed at ameliorating motor symptoms associated with DA deficiency, including levodopa, DA agonists, catechol-O-methyltransferase inhibitors, and monoamine oxidase-B inhibitors. 2,3 However, these therapies do not slow down disease progression and their efficacy decline over time, accompanied by severe side effects such as dyskinesia and on-off fluctuation. 4,5 A significant progress has been made in cell therapy for PD treatment in the last 30 years, with the grafted cell sources from fetal tissue to induced pluripotent stem cells. 6 Neural stem cells (NSCs) are considered to be the most promising source of transplanted cells due to their self-renewing and multipotent characteristics. 7 However, some issues still exist. One concern is that most in vivo transplanted NSCs are differentiated into glial cells rather than neurons. Another challenge is that only 5%-10% of the NSCs survive after transplantation due to the toxic effect of the inflammatory environment. 8 Previous studies have shown that the microglia activation and the associated inflammatory changes are considered as major pathogenic contributors to PD. [9][10][11] Microglia can produce both pro-inflammatory and neurotrophic cytokines in the central nervous system. 12,13 Therefore, a strategy biased toward beneficial microglia could be a promising therapeutic approach in PD.
Nuclear receptor-related factor 1 (Nurr1, also known as NR4A2) is an orphan nuclear receptor initially characterized as a transcription factor, which is critical for the development, differentiation, maintenance, and survival of DA neurons in the midbrain. 14 A reduction in the expression of Nurr1 in adult mice leads to a progressive loss of mDA neurons. 14 Conversely, overexpression of Nurr1 using a viral vector protects dopaminergic neurons against α-synuclein toxicity by restoring glial cell-derived neurotrophic factor (GDNF) levels. 15 In addition, Saijo et al 16

| Microglia cultures
Primary cultures for mixed astrocytes and microglia were derived from the cerebrum of SD rat pups on postnatal day 1 (PN1) using the protocol previously described. 18 Briefly, the cerebrum was removed and minced, and the cells were cultured in DMEM/high glucose containing 10% fetal bovine serum (FBS; HyClone, China). The cells were plated in 75-cm 2 T-flasks. The microglia were harvested by gentle shaking and collected for further use 12-14 days after initial seeding. inflammatory, microglia, neural stem cells, nuclear receptor-related factor 1, Parkinson's disease GFP (pCDH-copGFP-Nurr1) and RFP (pCDH-copRFP-Nurr1) were used for transfecting the NSCs and microglia, respectively.

| 6-OHDA lesion and amphetamine induced rotations
Male SD rats received unilateral stereotaxic injections of 6-OHDA (Sigma) into the median forebrain bundle as previously described 19 :

| Transplantation procedure and behavior test
Before transplantation, the neurospheres and microglia were har- Behavioral testing was carried out 3, 6, 9, and 12 weeks after transplantation. APO (Sigma) was subcutaneously injected at a dose of 0.25 mg/kg, and rotation was monitored for 30 minutes.

| Western blot analysis
After 12 weeks of surgery, the transplanted striatal tissues (n = 4 per group) were extracted in ice-cold RIPA lysis buffer. A total of 60 µg protein (bicinchoninic acid protein assay; Tiangen Biotech) from each group was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto the membrane using a semi- Millipore) for 2 hours at room temperature. Proteins were detected using the enhanced chemiluminescent reagent (Millipore). The relative levels of immunoreactivity protein were quantified using ImageJ software (NIH), and data were normalized to β-actin before statistical analysis.

| Reverse transcription polymerase chain reaction
Total RNA was extracted from tissues (n = 4 per group) using TRIzol-Universal reagent (Tiangen Biotech) and used for complementary DNA (cDNA) synthesis. Nurr1, TH, Pitx3, DAT, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control) cDNA fragments were amplified using the following primers: Nurr1 and GAPDH (forward 5′-TGCCTCCTGCACCACCAACT -3′, reverse 5′-CCCGTTCAGCTCAGGGATGA -3′). All transcripts were amplified by initial denaturation at 94°C for 3 minutes, 35 cycles of 94°C for 45 seconds, 58°C for 45 seconds, and 72°C for 1 minute, and a final extension at 72°C for 10 minutes. The polymerase chain reaction (PCR) products were subjected to 1% agarose gel electrophoresis, and the abundance of each mRNA was normalized to GAPDH using ImageJ software (NIH).

| Cell counting and statistical analysis
Immunoreactive cells were counted in 10-20 random areas of each culture coverslip using an eyepiece grid at a final magnification of 400×. Data were expressed as mean ± standard error of the mean of three independent experiments. For every figure, statistical tests were justified as appropriate. Statistical comparisons were determined using one-way analysis of variance followed by Bonferroni post hoc analysis using SPSS (Statistics 21; IBM Inc). A P value <0.05 was considered significant.
Then, these neurospheres were collected by centrifugation, resuspended in neural stem cell differentiation medium (DMEM/ F12, 1% FBS), and cultured for 7 days on polylysine-coated coverslips. After 7 days in culture, the cells derived from NSCs were stained with antibodies against Tuj1 and glial fibrillary acidic protein ( Figure 1B and 1C). In addition, TH immunoreactivity was detected in subpopulations of TuJ1+ neuronal cells ( Figure 1D). Importantly, these TH+ cells were considered as mature DA neurons, which were evidenced by the expression of DAT (DA transporter, Figure 1E).
Primary microglia cells were isolated after 14 days of mixed glial cell culture. The resting form of microglia was composed of long branching processes and a small cellular body. The ramified microglia could be transformed into the reactive state form in response to lipopolysaccharide (LPS)-mediated inflammatory environment. These cells were immunoreactive for CD11b (95%; Figure 1F).

| Nurr1 overexpression in NSCs and microglia
The green fluorescent protein and red fluorescent protein were    Figure 3E). Conversely, the results suggested that forced expression of Nurr1 in microglia led to a significant reduction in the expression of the pro-inflammatory cytokines, such as tumor necrosis factor-α(TNF-α) and interleukin-1β (IL-1β; Figure 3F).

| Transplantation of NNSC + NMG reversed motor behavior deficits in PD rats
Based on the in vitro results, the study next examined whether NSCs and microglia both with Nurr1 overexpression could improve the rotational asymmetry in PD rats. The rotational response to amphetamine was examined 3, 6, 9, and 12 weeks after transplantation ( Figure 4). Animals grafted with NSC, NNSC, NNSC + MG, or NNSC + NMG showed recovery 3 weeks after surgery from amphetamine-induced turning behavior, whereas the control (sham group) animals did not. However, in the NSC group, the rotation number reached a plateau in around the sixth week and even increased after it. Importantly, the rotation behavior in the NNSCs + NMG group significantly improved as the rotation number decreased over time.
Three rats were sacrificed because of sickness before the endpoint of the study. Both GFP and RFP derived from lentiviral vectors were still detectable under the fluorescence microscope; also, TH+ cells were observed in the striatum (Figure 9). These findings collectively indicated that co-transplantation of Nurr1-overexpressed microglial cells with Nurr1-overexpressed NSCs ensured a long-term significant outcome in PD rats via anti-inflammatory actions to improve hostile brain environments.

| D ISCUSS I ON
Stem cell-based cell replacement is a highly promising therapeutic approach because the main pathology of PD is selective degeneration of mDA neurons in the SN. However, the efficient differentiation F I G U R E 4 Amphetamine-induced rotation scores were analysed after cell transplantation. Each value depicts mean ± SEM of percent change in the score compared with the pretransplantation value. *P < 0.01 vs the control group (sham group), #P < 0.05 vs the NSC group, ≠P < 0.05 vs the NNSC group, and △P < 0.05 vs the NNSC + MG group, one-way analysis of variance followed by Bonferroni post hoc test F I G U R E 1 0 Schematic summary of the DA neuron survival and the neurotrophic effects of Nurr1-Microglia and neural stem cell co-grafted in PD treatment. Activated microglia produce pro-inflammatory cytokines that contribute to DA neurons death. Nurr1overexpressed microglial cells co-grafted with Nurr1-overexpressed NSCs promote the survival of transplanted NSCs, and DA neuron differentiation. As result, the abnormal behaviors in PD rats are improved by correcting the inflammatory host brain environments change suggested that the outcomes in these experimental groups improved for the presence of Nurr1 overexpression. Furthermore, the increase in the expression levels of Pitx3, TH, DAT, and Nurr1 was further confirmed using RT-PCR and Western blot analyses ( Figure 6).
They were all DA neuron-related transcription factors, indicating that the number of DA neurons obviously increased in the NNSC + NMG group. Next, the present study used immunofluorescence to investigate whether forced expression of Nurr1 reduced the activation of microglia in the striatum of PD rats. The results showed that the expression level of Iba1+ cells in the NNSC + NMG group decreased 12 after transplantation compared with the other groups (Figures 7 and 8).
Moreover, this study found that most implanted cells were localized in the transplant area, and only a few of them were observed in the striatum of PD rats in the NNSC + NMG group after months (Figure 9).
Based on the results and previous studies, the molecular mechanisms underlying the neuroprotective effects of Nurr1 on PD included the reduction of inflammatory factors and the secretion of neurotrophic factors and other cytokines, such as SHH and FGF8. 21,33

| CON CLUS ION
In conclusion, the aforementioned in vitro and in vivo findings demonstrated that the overexpression of Nurr1 promoted the differentiation of NSCs into DA neurons, increased the number of TH+ cells in the striatum, and reduced the number of Iba1+ cells, resulting in an improvement in the rotation behavior. Therefore, transplantation of Nurr1-overexpressing NSCs and microglia, which aims to improve the inhospitable host brain environments, has great potential for treating PD.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C A L A PPROVA L
This study was approved by the institutional review board and the Animal Care and Use Committee of Kunming Medical University.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA ACCE SS I B I LIT Y
All data generated or analyzed during this study are included in this published article.