Therapeutic effect of TO901317 on 6-OHDA-induced Parkinson rats in vitro and in vivo

Background: Stem cells from different sources could differentiate into dopamine-producing cells and ameliorate behavioral deficits in Parkinsonian models. Especially, human bone marrow mesenchymal stem cells (hBMSCs) have many advantages without ethical dispute. Liver X receptor s (LXRs) are involved in the maintenance of the normal function of the central nervous system myelin. We have reported the induction of cocktail-induced da phenotypes from adult rat BMSCs by using sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF) and TO901317 (agonist of LXRs) with 87.42% of efficiency in 6 days of period of induction. But the previous work did not verify whether the induced cells had the corresponding neural function. Methods: In this study, we demonstrated that TO901317 could promote the differentiation of hBMSCs into dopaminergic neurons. Neuronal markers (Tuj1, Neun and Nestin), dopamine neuron markers (tyrosine hydroxylase, TH), LXRa and LXRb were detected by immunofluorescence. RT-qPCR was used to measure the mRNA expressions of adenosine triphosphate-binding cassette transporter A1 (ABCA1). Western Blotting detected the changes of LXRa, LXRb and TH expression. Results: TO901317 significantly enhanced the differentiation from hBMSCs to DA neurons. Only the LXR+GF group released dopamine by the result of enzyme linked immunosorbent assay (ELISA). Compared with the control group and GF group, the optimal time for differentiation of hBMSCs treated by 0.5mM TO901317 combined with GF was six days. And the maximum induction efficiency was 91.67%. After transplanting induced-cells into Parkinson's disease rats, the symptoms of Parkinson's rats decreased, and the number of dopamine neurons increased in the substantia nigra and striatum. Conclusions: TO901317 promoted differentiation of hBMSCs into dopamine neurons may be related to activation of LXR-ABCA1 signaling pathway. These data suggest that TO901317

patient-specific and disease-specific neurons, especially as, in theory, this approach would avoid many of the ethical issues associated with using ESCs 10,11 . Cell replacement therapy is derived from the patient's own cells, avoiding immune rejection. In autologous transplantation, it is necessary to establish iPSCs from each patient, and current technical operations take much time and high cost, so it is difficult to spread to general treatment. In addition, the use of iPSCs with the patient's own genetic factors, the sensitivity of the disease may be high. It also has the risk of introducing cancer cells 12 . Specifically, iPSCs often obtained chromosomal abnormalities, with gains or losses of whole chromosomes 13 . Different from these stem cells, BMSCs come from patients themselves without ethics dispute. Especially, BMSCs have multi-directional differentiation potential 14 , own low risk of tumorigenesis 15 and are rich in source, easy to extract, separate and purify. Many studies in vitro and preclinical strongly proved the therapeutic potential of BMSCs when be applied as a treatment for different pathological conditions 16,17 .
There have been different methods involved in the differentiation of BMSCs, including cell growth factors 18,19 , chemical inducer 20 and lentiviral transduction 21 . Currently, in vitro induction of stem cells using growth factors with sonic hedgehog (SHH) and fibroblast growth factors (FGFs), succeeded in inducing adult human BMSCs into DA neurons with 67% of efficiency in 12 days 22,23 . This is the current maximum induction efficiency and the shortest induction time.
Liver X receptors (LXRs) include LXRα and LXRβ. It is the member of the nuclear receptor supergene family of ligand-activated transcription factors and is a major regulator of lipid metabolism 24 . It play a key role in the regulation of cholesterol and fatty acid homeostasis 25 . LXRs is also essential for central nervous system (CNS) 26,27 . The loss of LXRβ in mice affected the formation of progenitor cells and granule cell differentiation, and then leading to hypoplasia of the dentate gyrus 26,27 . It has been found that LXRs played crucial roles in regulation of genes related to CSF production and structural integrity of choroid plexus 29 . LXRα and LXRβ are involved in the processes of myelination and remyelination 30 . Activation of LXRα and LXRβ can promote the regeneration and survival of motor neurons 31 . Therefore, LXR agonists activate LXR target genes, plays therapeutic role in different neurodegeneration animal models 32-34 . Our previous work and others have found the induction of cocktail-induced da phenotypes in adult rat BMSCs by using SHH, fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF) and TO901317 with 87.42% of efficiency in 6 days of period of induction 35 . LXR agonist significantly shortened induction time and improved induction efficiency compared to methods which had been reported. But these previous work did not investigate whether induced-cells released dopamine and had the corresponding dopaminergic neurons. We did not sure if the induced cells have a therapeutic effect on Parkinson's disease.
In this study we investigated the effect of TO901317 on the differentiation of hBMSCs into dopamine neurons. We also explored whether induced-cells had dopamine neuronal function and the possible mechanism. Finally, we transplanted the induced-cells into PD rats to observe the therapeutic effect.

Animals
Sprague-Dawley (SD) rats were accommodated in the barrier housing facility, in keeping with the national standard of "Laboratory Animal-Requirements of Environment and Housing Facilities." The care of the laboratory animal and the animal experimental operation conform to the "Chongqing Administration Rule of Laboratory Animal." The experimental procedures were approved by the animal laboratory administrative center and the institutional ethics committee of Chongqing Medical University. All male SD rats were weighted 220g-250g. To establish the rat model of PD, 30 male rats received a 6-OHDA lesion of the medial forebrain bundle (MFB) on the right side and 10 rats were as control group only injected with solvent which was used to dissolve 6-OHDA 36 .

Differentiation of hBMSCs
Human BMSCs were divided into 3 groups and plated onto 24 well plates where each plate contained 2.0×10 4 cells and cells were cultured at 37°C with 5% CO2.

2.
Explore the best time to add TO901317 during growth factor induction period: On the basis of GF, TO901317 were added on the first day, the third day, the sixth day, and the ninth day, respectively, to induce differentiation for 12 days.
3. Explore the induction time of the TO901317 in combination with GF: According to the result of (a) (b), 0.5 μM TO901317 was added to the culture medium, and the cell morphology was observed every 3 days.

Cell Counting kit-8 assay
A Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay was used to test the growth rate by following the manufacturer's instructions. 3000 cells/well of hBMSCs were cultured into a 96-well plate in every group. At 3-day intervals, the growth rates of cells in each group were measured by application of CCK-8 kit and optical density (OD) was determined at 450 nm using a microplate reader (Thermo Scientific, USA).

Parkinson's disease rats
All male SD rats were fed to 220-250g. Rats were intraperitoneally injected with 0.5 mg/kg apomorphine. Rats without rotation behavior were selected to establish PD model. To generate unilateral PD models, 6-OHDA was injected into MFB in 30 rats 37 .

Immunofluorescence
In brief, cells of each group were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton-

Enzyme-Linked Immunosorbent Assay (ELISA)
To investigate whether cells in each group release dopamine. Culture supernatant and cells of control group, GF group and LXR+GF group were collected. Cells were added with PBS, then grinded on ice.
The mixture was centrifuged at 3000g for 20min at 4°C and collected the supernatants. Dopamine was detected by ELISA kits (n=6) (Mei biao, Jiangsu, China). The samples and standards were tested according to the instructions.

Western Blotting Test
Cells were plated on six-well plate for 24 hours with 10% FBS, then grown in induction medium for six days to be used for preparation of whole cell extract. After removing the media, cells were washed three times with PBS. Then cells in each well were added 150ml lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) and cracked on ice for 30 minutes. The mixture was centrifuged at 12,000 g for 20 min at 4°C and collected the supernatants. The protein concentration was determined by BCA Protein Assay Kit (Beyotime, China). The protein was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, United States). The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and then incubated with specific primar antibodies, TH, 1:500 (Abcam, Cambridge, UK); LXR α receptor, 1:500 (Abcam, Cambridge, UK); LXR β receptor, 1:500 (GeneTex, United States) were included and overnight at 4℃. The membranes were rinsed three times in TBST and incubated with HRP conjugated secondary antibodies at room temperature for 1h. Then after washed three times in TBST, protein signals were visualized by ECL (Bio-Rad, United States).

Quantitative real-time PCR (qPCR)
Total RNA was isolated from the cells in control group, GF group and LXR+GF group by Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer's protocol. Then mRNA was subjected to reverse transcription using HiScript Q Select RT SuperMix (Vazyme, Nanjing, China). SYBR Green II (Biomake, USA) incorporation method was used to detect the amount of mRNA. Negative controls were used as no template cDNA reactions and melting curves were used to confirm the results. The results were normalized using GAPDH concentration of each sample.

Histopathological Examination
Hematoxylin and eosin (HE) staining was performed to show pathological histological damage in the substantia nigra pars compacta (SNc) and striatum. The rats of control group, 6-OHDA group and 6-OHDA+Cells group were anesthetized with sodium chloral hydrate and perfused with PBS, and then perfusion with 4% paraformaldehyde. After that, the brains were dehydrated in a graded series of alcohols and embedded in paraffin. A series of 5-μm-thick sections were cut from the brain. Finally, the sections were stained with HE reagents for histopathological examination.

Statistical analysis
All results were expressed as the means ± standard deviation (SD) and the statistical significance of differences was analyzed by GraphPad Prism (GraphPad Software, La Jolla, CA, USA). For the comparison of multiple groups, statistical analysis was performed using one-way analysis of variance (ANOVA) with Bonferroni's posthoc test. Probability values less than 0.05 (p < 0.05) were considered to be statistically significant.

Culture of hBMSCs
hBMSCs were purchased in Cyagen Biosciences. Cells exhibited a long fusiform and vortex arrangement (FIGURE 1).

Effect of different concentrations of TO901317 on cell survival
CCK8 kit was used to detect the survival rate of cells in control group, GF group and LXR+GF group. In addition, the concentration of TO901317 in the LXR+GF group was given to 0.125mM, 0.25mM, 0.5mM, 1mM and 2mM respectively. After three days, the survival rate of cells in each group did not show significant difference (FIGURE 2A). The survival rate significantly increased in GF group and LXR+GF group compared with control group when cells were cultured for six days, nine days and twelve days. Interestingly, there was no significant difference in cell survival rate when cells were cultured with different concentrations of TO901317 (FIGURE 2).

Determination of the concentration of TO901317
Immunofluorescence was used to detect the expression of neuronal markers (Neun, Nestin and Tuj1) and dopamine neuron markers (TH).
In this study, TO901317 was used of five concentrations of 0.125mM, 0.25mM, 0.5mM, 1mM and 2mM. We found that when GF combined with 0.5mM TO901317, the number of TH + positive cells reached the maximum ( ** P < 0.01) (FIGURE 3).

Explore the adding time of TO901317
In this study, we used 0.5mM TO901317 in combination with GF to induce hBMSCs into dopaminergic neurons. On the premise of GF addition during 12 days, TO901317 was added on the first day, third day, sixth day, and ninth day. Expression of TH (Cy3, red) and Tuj1 (Alexa Fluor 488, green) were determined by immunofluorescence. The results showed that the expression of TH was the highest when TO901317 was added on the first day (FIGURE 4).

Explore the time period induced by TO901317
Immunofluorescence was used to detect the expression changes of Tuj1 and TH after three days, six days, nine days and twelve days of induction period. The control group only expressed Tuj1 without TH. While GF group and LXR+GF group expressed Tuj1 and TH simultaneously (FIGURE 5A). In the LXR+GF group, TH + cells reached maximum after six days of induction (FIGURE 5B). Expression changes of TH was detected by Western blotting in Control group, GF group and LXR+GF group.
Compared with control and GF groups, the expression of TH was significantly increased in LXR+GF group (FIGURE 5D, E). Cells showed typical neuronal morphology with extended long cell processes and enlarged cell bodies in GF group and LXR group under the bright field (FIGURE 5C). In particular, in the LXR+GF group, the morphology of the neurons expressed by the cells was more obvious (FIGURE 5F).

Dopaminergic neuron properties of inducing cells
Nestin, a neuroectodermal marker, seems to be a prerequisite for acquisition of the aptness to progress towards the neural lineage 38, 39 . The cells in the control group probed with nestin and neun antibodies were negative and just revealed nuclear staining DAPI. Both GF group and LXR+GF group, cells showed staining with neun and nestin (FIGURE 6A, 6B).
To study whether differentiated hBMSCs release dopamine, a dopamine kit was used. Cells in control group and GF group did not secrete dopamine. Only the LXR+GF group secreted dopamine ( FIGURE   6C).

The role of LXRs in cell differentiation and its possible mechanism
Expression of LXRα and LXRβ between control group, GF group and LXR+GF group did not show significant difference (FIGURE 7A, B, E, F). Western blot result showed that expression of LXRα and LXRβ were decreased significantly in LXR+GF group compared to control group and GF group (FIGURE

7C, D, G, H).
Adenosine triphosphate-binding cassette transporter A1 (ABCA1) is the target gene of LXRs. The lack of ABCA1 leads to transport disorders of central nervous system cholesterol, which in turn leads to defects in neuronal structure and function. Compared with control group and GF group, the mRNA of ABCA1 of LXR+GF group was significantly elevated (FIGURE7I).

Establishment of Parkinson's disease model
All SD rats (220g-250g) received intraperitonal injection of apomorphine dissolved in sterile (0.5mg/kg). Choosing rat without rotation behavior received a 6-OHDA lesion of the MFB 37 . The 6-OHDA were dissolved in saline with 0.02% ascorbic acid solution containing. Briefly, rats were anesthetized with chloral hydrate (4% chloral hydrate and 96% saline solution) by intraperitoneal injection. The MFB was targeted with an injection of 6-OHDA with a total amount of 14 μg. The lesion to stereotaxic coordinates were adjusted to the age and weight of the animals with the help of brain stereotaxic instrument (RWD, Shenzhen, China). After two weeks during one month, to determine whether the models were successful, we used apomorphine to induced rotation and behaviour was recorded over a period of 90 min. Judging that the model is successful is that the number of rotations to the healthy side is greater than 7 rotations per minute (FIGURE 8).

The vivo effects of inducing cells
Four weeks after 6-OHDA infusion, rats were divided randomly into three groups as follows:the control group as solvent group (n=10), the model group as 6-OHDA group (n=10) and the therapy group as 6-OHDA +cells group (n=20). 1×10 5 induced dopaminergic neurons in 2 μL transplantation buffers (phosphate buffer saline) were injected into the right side of the SNc (A/P -4.6 mm, M/L −2.2 mm, D/V −7mm). The 6-OHDA group was only injected transplantation buffers at the same place as 6-OHDA+Cells group.
After four weeks, the apomorphine-induced contralateral rotation test was performed. the 6-OHDA + In the reported studies, many methods were adopted to drive BMSCs to differentiate into DA neurons. ABCA1 affects cognitive function, leading to amyloid-beta (Aβ) production and apoptosis and promote neurorestoration 60-62 .
In the present study, our aim was to explore the effects of TO901317 on the differentiation of hBMSCs into dopamine neurons. We found that the growth rate of cells in GF group and LXR+GF group was initially increased and later decreased with the extension of induction time compared to control group. But the growth rate of cells in LXR+GF group was not different with GF group. We hypothesized that TO901317 had little or no effect on cell proliferation, and TO901317 may have a special effect on cell differentiation. Some studies have explored the effect of GF on inducing BMSCs differentiation into dopaminergic neurons, but the result were designated DA neuronal progenitors without neuronal function 63, 64 . Our results showed that hBMSCs treated with GF alone or TO901317 combination with GF leaded to directed neuronal differentiation. TO901317 could promote differentiation of hBMSCs into dopaminergic neurons in cooperation with GF. The maximum induction efficiency was 67%, and the shortest induction time was 12 days when hBMSCs treated with GF alone.
While the maximum induction efficiency was 91%, and the shortest induction time was 6 days when hBMSCs treated with 0.5μM TO901317 in cooperation with GF. This greatly improved the efficiency of induction. Cells in GF group and LXR+GF group showed neun and nestin positive. The result of ELISA revealed cells only in LXR+GF treated secreted dopamine. This suggested that TO901317 could promote the maturation of cellular functions. All of these results together indicated that simultaneous addition of TO901317 and GF could significantly improve induction efficiency and shorten induction period of hBMSCs differentiation into DA neuron-like cells and suggested that LXR may be involved in the mechanisms of hBMSCs differentiation into DA neuron-like cells.
The lack of ABCA1 leads to transport disorders of central nervous system cholesterol, which in turn leads to defects in neuronal structure and function 65 . Our study found that ABCA1 mRNA significantly elevated and LXRs obviously decreased in LXR+GF group compared to GF group. Differentiation of hBMSCs into dopaminergic neurons may be related to LXR-ABCA1 pathway.   Figure 1 hBMSCs were observed at inverted microscope and showed a fibroblastoid cell profile and produced cell colonies with unique vortex shape. (D) Culture for 12 days. Data were expressed as mean ± SD (n = 6). *P < 0.05, **P< 0.01, ***P< 0.001, compared with control group.      Data were expressed as mean ± SD (n = 3). *P<0.05, compared with 6-OHDA group. (F) The results of apomorphine-induced contralateral rotations from each group. Data were expressed as mean ± SD (n = 6). ***P<0.001, compared with model group.