Human Amniotic Epithelial Cells Recover Mouse model of Parkinson’s Disease mainly by NeuroprotectiveAnti-oxidative and Anti-inflammatory factors CURRENT STATUS: UNDER REVIEW

Objectives: Human amniotic epithelial cells (hAECs) have been reported to have neuroprotective roles in neurological diseases including Parkinson’s Disease (PD) in animal models. However, the mechanism is not fully understood. Materials and method : Firstly, hAECs were transplanted into the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( MPTP )-induced PD mice. And the motor deficits were tested by Rotarod test. Secondly, injury severity of nigral dopaminergic neurons in substantia nigra and striatal axon fibers in PD mice was estimated by immunochemistry with tyrosine hydroxylase antibody. Thirdly, neuroinflammation was measured by microglial activation and the level of inflammatory factor levels. Lastly, the oxidative stress was tested by the level of reactive oxidative species (ROS) levels. In vitro, we examined a full spectrum of soluble proteins of hAECs by Raybiotech human Antibody Array 507. Besides, antibody neutralization experiments were used to determine the role of many factors on dopaminergic neurons. Results: hAECs significantly attenuated the motor deficits of PD mice. Moreover, the grafts prevented the loss of nigral dopaminergic neurons, promoted the outgrowth of their neurites and striatal axon fibers in PD mice. More importantly, decreased microglial activation, inflammatory factor levels and MPTP-induced excessive ROS levels were also observed in hAEC-treated PD mice. In vitro, analysis of an antibody array of 507 soluble target proteins using hAEC-CM revealed that the levels of many neurotrophic factors, growth factors neuronal cell adhesion molecule ( NrCAM )and antiinflammatory factors were evidently high. In addition, antibody neutralization experiments showed that many of them contributed to the survival and growth of dopaminergic neurons and the outgrowth of their neurites. Conclusion: Our study indicates that the neuroprotection effects by hAECs grafts are achieved not only by neuroprotection and neurite outgrowth-promoting activities, but also by anti-inflammation and anti-oxidation functions in the microenvironment of the damaged dopaminergic neurons, so to facilitate the restoration of neurological functions. leukocyte antigen HLA-DR. The results indicated that hAECs isolated have a high purity and are useful for the following experiments.

Introduction from eBioscience, San Diego, USA). Upon completion of washing with PBS, labeled cells were resuspended and at least 10 5 events were acquired by using a BD Accuri TM C6 Flow Cytometer (BD, NJ, USA).

Transplantation of hAECs into PD Mice Induced by MPTP
A repeated MPTP (30mg/kg, intraperitoneal injection for six consecutive days) were administered to C57BL/6 mice to induce PD mouse model. Mice treated with PBS were used as a control (CON).
Recipient mice were divided into three groups : "CON" group (n≥8), "MPTP+PBS" group (n≥8) and "MPTP+hAECs" group (n≥8). In the "MPTP+hAECs" group, mice treated with MPTP received unilateral transplantation of hAECs at day 7. hAECs were digested and prelabeled with PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma-Aldrich). Cell suspension (3ml, 1×10 6 cells) in PBS was stereotaxically injected into the unilateral striatum of recipient mice according to the following coordinates: 0.8 mm posterior to bregma; 2.0mm lateral to bregma; 3.0 mm ventral from the dural surface with the tooth bar set at zero. PBS was injected in a manner as above.

Rotarod test
The rotarod behavior test was measured using a rotarod device (RWD life Science, Shenzhen, China).
The parameters of the rotarod system include starting speed, acceleration and the highest speed (2rpm, 1.5rpm/s, 50rpm). Every mouse underwent three consecutive trials and the longest latency time for each mouse to fall off the rotating rod for the last two trials was measured at week 1 and week 2 after transplantation of hAECs. The rest period between each trial was 30 min.

Immunohistochemistry and Immunofluorescence Staining
Four weeks after transplantation, mice were perfused and the brain were fixed and cut into a thickness of 35 mm coronal sections using a freezing microtome (Leica, Wetzlar, Germany). For the dopaminergic neurons in the substantia nigra (SN) of a mouse, 24 consecutive coronal sections (35µm) covering the entire SN were obtained [29]. For immunohistochemistry, the sections were incubated with primary antibodies against TH (1:2000, Abcam, Cambridge, USA) or Iba-1 (1:500, Wako, Japan). After washing with PBS, the sections were incubated with the biotinylated secondary antibody anti-rabbit IgG (1:200, Vector, Peterborough, U.K.). Then the sections were visualized using a Vectastain ABC Kit (Vector) at RT 30 min and developed with 3,3'-diaminobenzidine at RT. The imagines were visualized by using an inverted microscope. Manual counts were performed to estimate the number of surviving TH neurons in SN of mice and Image J software were used to quantify the density of TH immunostaining in the striatum of mice.
For immunofluorescence, the cells were incubated overnight at 4℃ with the primary antibodies as follow: rabbit anti-TH (1:300), mouse anti-human Nuclei (1:400, Millipore, Billerica, MA). After washed with PBS, the cells were then incubated by the corresponding conjugated secondary antibodies: Alexa Fluor 488 and Alexa Fluor 594. Cell nuclei were labeled with DAPI (Sigma). The imagines were visualized by using an inverted fluorescence microscope or ZEISS confocal microscope.

In situ visualization of ROS production
In situ visualization of reactive oxidative species (ROS) production was assessed by 2',7'dichlorodihydrofluorescein diacetate (H2-DCFDA, Life Technology) histochemistry according to previous reports (Quick and Dugan, 2001;Wu et al.,2003). The intensity of fluorescence was determined by Image J. The obtained values were presented as a percent of the control (MPTP group).

ELISA Assay
The concentrations of IL-1b and TGFa in the serum of mice were measured by using mouse IL-1b and TGFa ELISA kits (eBioscience).

Primary Mesencephalic Neuron Cultures and Co-cultures with hAECs
Primary mesencephalic neuron-glia cultures were prepared from the brains of embryonic day (E) 13.5 wild-type C57BL/6 mouse embryos according to previously described protocol [30]. Mesencephalic cells were cultured in the neural basal medium containing 1% B27, 0.5mM L-glutamine ( all from Life Technology), then mesencephalic neuron-glia cultures were treated with 2.5mg/ml cytarabine (Sigma Aldrich) for two days to remove glial cells, subsequently, the cultures were treated with 20μM 1methyl-4-phenylpyridine (MPP + , Sigma-Aldrich) for 24h at 37℃. Then hAECs were digested and cocultured with above mesencephalic neurons

Analysis of Soluble Factors of hAEC-CM
When hAECs were cultured to 90% confluence, hAECs were rinsed with PBS and continued to culture in the basal medium for 24h at 37°C. Then the supernatant was used as the conditioned medium (CM) of hAECs (hAEC-CM) and kept at -80℃. The CM of adult foreskin fibroblasts (hEF) (hEF-CM) used as a control was harvested as above. To test the neurotrophic factors and growth factors in the hAEC-CM and hEF-CM, protein antibody array was performed with Raybiotech L-series human Antibody Array 507 (Raybiotech, Atlanta, USA). The expression levels of 507 human target proteins, which includes cytokines, chemokines, adipokines, growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules and other proteins, can be simultaneously detected. The procedure was done according to the manual of manufacture. Finally, the glass slide is dried, and fluorescence signals were scanned with a GenePix 4000B (Axon Instruments, GenePix version 5.0). For each array, protein intensity values were background subtracted, scaled by the internal control, and floored at 1 unit.

Statistical Analysis
Data were presented as mean ± SEM (n≥3 experiments), and statistical significance of effects between a control and treatment group was determined using Student's t test, P<0.05 (*), P<0.01 (**), and P<0.001 (***). Microarray data were statistically analyzed with the significance analysis of microarrays (SAM) method.

Cultures and Characterization of hAECs
In order to establish the hAECs cultures, hAECs were isolated according to a modified protocol as previously described [28]. hAECs were derived from the epithelium layer of placenta and cultured in DMEM/F12 containing 10% FBS. In the cultures, hAECs displayed an epithelial morphology (Fig.S1A).
Moreover, hAECs expressed high levels of Epcam, CD49f, CD29 and CD73, but much less levels of CD31, CD34, CD45 and human leukocyte antigen HLA-DR. The results indicated that hAECs isolated have a high purity and are useful for the following experiments.

Deficits of PD Mice
To determine if hAECs that we prepared attenuate MPTP-induced neurodegeneration and ameliorate behavioral deficits of PD mice, PD mice were induced by intraperitoneal injection of MPTP for consecutive six days. Then hAECs were transplanted into the unilateral striatum of PD mice at day 7  (Fig. 1D). Furthermore, the density of the striatal axon fibers in the PD mice was enhanced after hAECs transplantation (Fig. 1E, 1F). Remarkably, the hAECs grafts ameliorated MPTP-induced motor deficits, as measured by the rotarod test at 2 weeks after transplantation (Fig.   1G), but hAECs did not display an ameliorate effect at 1 week post-grafting (data not shown). Taken together, the results suggested that hAECs attenuated MPTP-induced motor deficits and promoted the survival of dopaminergic neurons and outgrowth of their neurites and projected striatal axon fibers.

Survival of Transplanted hAECs
To evaluate the survival of transplanted hAECs, they were prelabeled with PKH26 red fluorescent dyes and then transplanted into unilateral striatum of MPTP-induced PD mice. As shown in Fig. 2, the grafts could survive at least four weeks after transplantation, as demonstrated by human nuclei (hNuclei)immunoreactive cells observed in the unilateral striatum of PD mice. Interestingly, the number of hNuclei positive cells in the striatum at 2 weeks post-grafting were higher than that at 4 weeks postgrafting (Fig 2), which indicated that hAECs were not overgrown. Surprisingly, double positive for hNuclei and TH double were rarely been observed (data not shown), suggesting that hAECs are difficult to differentiate into dopaminergic neurons 4 weeks post-grafting.

hAECs Promote Survival and Neurite Outgrowth of Mesencephalic Dopaminergic Neurons
NrCAM compared to that of IgG group. Quantification revealed a lower percentage of the dopaminergic neurons bearing neurites, and significantly shorter neurites length and fewer branching points per dopaminergic neuron in the present of neutralizing antibodies against these factors compared to those of IgG group (Fig.4E). In addition, the survival of lesioned dopaminergic neurons was evidently reduced in the present of neutralizing antibodies against anti-inflammatory factors IL-1raand IL13, but not with anti-inflammatory IL-10. In particular, the branching points per dopaminergic neuron was also much lower compared to that of IgG group in the present of neutralizing antibodie against IL-1ra( Fig.4D and 4E). Collectively, hAECs have neuroprotective effects on PD mice attributed to the paracrine factors including neurotrophic factors, growth factors, NrCAM, anti-inflammatory factors.

hAECs Inhibits Microglial Activation and Neuroinflammation of MPTP-induced PD mice
Neuroinflammatory processes including activated microglial and increased concentration of proinflammatory factors play an important role in the progression of PD [33,34]. Here, activated microglia displaying an amoeboid-cell body were evident, and elevated concentration of IL-1b and TGFa was also observed in MPTP-induced PD mice (Fig. 5). Interestingly, 4 weeks after transplantation of hAECs into the PD mice, hAECs attenuated the microglial activation in the SN of PD mice, as validated by the morphology change of microglial cells from a larger and amoeboid cell body with a few ramified patterns to a round cell body with many ramified patterns similar to that in the CON group (Fig. 5A). Besides, the levels of pro-inflammatory cytokines IL-1b and TNFa in the serum of PD mice were also decreased in the MPTP+hAECs group than those in the MTPT+PBS group (Fig. 5B).
Consistently, Fig. 4D, 4E also showed that the majority of anti-inflammatory cytokines such as IL-1ra, IL-10, IL-13 in the hAEC-CM were richer and promoted the dopaminergic neuronal survival. Taken together, the data revealed that hAECs play an anti-inflammatory role by inhibiting microglial activation and secreting some anti-inflammatory factors.

hAECs Attenuates MPTP-induced Oxidative Stress
Recent studies suggest that oxidative stress contribute to the loss of dopaminergic neurons in the pathogenesis of PD [35]. MPTP has been showed to cause the production of excess levels of reactive oxygen species (ROS) in a mouse models [36]. To measure the MPTP-elicited ROS level in the SN of PD mice, we co-stained brain slides with TH antibody and oxidant-sensing fluorescence probe H2-DCFDA. Fig.6A shows MPTP induced the production of a great deal of ROS, as evidenced by a high green fluorescence density in the SN of the mice of MTPT group compared to those of normal group (no MTPT).Interestingly, hAECs significantly reduced MPTP-elicited oxidative stress in the SN (TH positive area) of PD mice based on low green fluorescence density in MPTP+hAECs group compared to that of MPTP group (Fig.6A). Quantification revealed that the relative percentages of green fluorescence density in SN of PD mice in MPTP+hAECs group compared to that of MTPT group were evidently reduced (20.34%±5.85%) (Fig. 6B). Thus, the results indicated that hAECs grafts strikingly reduced MPTP-induced oxidative stress.

Discussion
Although previous studies reported that hAECs prevent nigral dopaminergic neurons from degeneration, and ameliorate behavioral deficits in PD rat models [16, 26, 27, 37], the mechanism for the neuroprotective effect of hAECs on the PD model remains unclear. In particular, regarding for PD pathology, whether there are progressive steps involved, such as an initial loss of small percentage of neurons and a subsequent failure of self-repair and death of these neurons is vague. In this study, we not only demonstrated that hAECs grafted into the striatum of PD mice reduce the loss of nigral dopaminergic neurons and improve the rotarod behavior of PD mice (Fig. 1), but also showed that the hAECs grafts promote the outgrowth of their neurites and striatal axon fibers in PD mice (Fig. 1D-G).
Therefore, hAECs inhibit the degradation of nigral dopaminergic neurites and striatal axon fibers projected by dopaminergic neurons contributing to the self-repair of the partially damaged dopaminergic neurons. Consistent with this notion, it is worth mentioning that Richard Burke et al reported that at the onset of PD symptoms only approximately 30% of SNpc dopamine neurons are lost, much less severe than the extent of the striatal axonal degeneration, indicating that the majority of dopamine neurons are still viable at this time point [38]. In other words, striatal axonal degeneration is one of the earliest features of PD pathology, which is then followed by neuronal death in the SNpc. Therefore, the present study provides both in vitro and in vivo evidence that prevention of neurite and axonal degeneration and promotion of self-repair of the partially damaged brain injury mice and decreases the activated microglia cells and astrocytes contributing to the reduction of neuroinflammatory response [45]. Additionally, Sachiko Tanaka found that LPS induces microglial activation and PD-like behavior impairment in both wild-type and TNFα KO mice, but not IL-1 KO mice. It is also proposed that IL-1 is essential in the initiation of neuroinflammatory response and neurodegeneration change that occur in PD [46]. Hence, IL-1ra likely plays a key role in reducing neuroinflammatory response in PD. Based on these data, it is reasonable to propose that hAECs exert anti-neuroinflammation effects in PD animal model by secreting IL-1ra and other anti-inflammatory factors, resulting in a decrease in the inflammation-mediated neurodegeneration in PD.

Conclusion
The present study shows that hAECs grafts not only promote the survival of dopaminergic neurons but also the outgrowth of their neurites in PD mice. More importantly, a protein antibody array assay gives a full spectrum of relative level of 507 paracrine factors and reveals that many neurotrophic improves motor function in an animal model of Parkinson's disease. Brain, 2008.  Survival of the hAECs grafts. The grafts were immunostained with human Nuclei (hNuclei) and TH at 2 weeks (2W) and 4 weeks (4W) post-grafting. hAECs were labeled with PKH26.
B. The concentrations of IL-1β and TGFα in the serum of mice in different groups were measured by ELISA assay. Con represents normal control.

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