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Keywords:

  • mesenchymal stem cell;
  • neuroprotection;
  • Parkinson's disease

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Result
  5. Discussion
  6. Acknowledgements
  7. Conflicts of Interest
  8. References
  9. Supporting Information

Parkinson’s disease is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. We investigated whether cell therapy with human mesenchymal stem cells (hMSCs) had a protective effect on progressive dopaminergic neuronal loss in vitro and in vivo. In primary mesencephalic cultures, hMSCs treatment significantly decreased MG-132-induced dopaminergic neuronal loss with a significant reduction of caspase-3 activity. In rats received systemic injection of MG-132, hMSCs treatment in MG-132-treated rats dramatically reduced the decline in the number of tyrosine hydroxylase (TH)-immunoreactive cells, showing an approximately 50% increase in the survival of TH-immunoreactive cells in the substantia nigra compared with the MG-132-treated group. Additionally, hMSC treatment significantly decreased OX-6 immunoreactivity and caspase-3 activity. Histological analysis showed that the number of NuMA-positive cells was 1.7% of total injected hMSCs and 35.7% of these cells were double-stained with NuMA and TH. Adhesive-removal test showed that hMSCs administration in MG-132-treated rats had a tendency to decrease in the mean removal time. This study demonstrates that hMSCs treatment had a protective effect on progressive loss of dopaminergic neurons induced by MG-132 in vitro and in vivo. Complex mechanisms mediated by trophic effects of hMSCs and differentiation of hMSCs into functional TH-immunoreactive neurons may work in the neuroprotective process.

Abbreviations used
BSA

bovine serum albumin

FACS

Fluorescence-activated cell sorting

MPTP

1-methyl-4-phenyl-1,2,3,6-Tetrahydropyridine

hMSCs

human mesenchymal stem cells

PBS

phosphate-buffered saline

PD

Parkinson’s disease

SDF-1α

stromal cell-derived factor-1α

TH

tyrosine hydroxylase

Parkinson’s disease (PD) is a chronic neurodegenerative disease characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies, proteinaceous inclusions that contain α-synuclein, synphilin-1, components of the ubiquitin proteasomal pathway, and parkin in the substantia nigra (SN) (Moore et al. 2005). Recently, various genes responsible for familial PD have been identified and these have led to an increased understanding of its molecular pathogenesis (Healy et al. 2004; Morris 2005). Of those, mutations in the parkin gene are relatively common in familial PD, being found in 50% of familial early-onset cases compatible with recessive inheritance (Healy et al. 2004; Morris 2005; Schrag and Schott 2006). Parkin has E3 ligase activity, an important part of the cellular machinery that covalently tags target proteins with ubiquitin through the ubiquitin-proteasome system (Hattori and Mizuno 2004). Thus, loss of parkin function results in improper targeting of its substrates, leading to inadequate protein degradation and consequently unfolded protein accumulation.

Recently, McNaught et al. (2004) reported that systemic injection of a proteasome inhibitor induced a chronic progressive neurodegenerative disorder in rats that closely recapitulates the features of PD in pathological, neurochemical and behavioral aspects. Furthermore, they suggested that this model could be a useful tool for testing putative neuroprotective therapies for PD. There is a variety of proteasome inhibitors, including lactacystin, PSI, epoxomicin and MG-132. These proteasome inhibitors consistently caused a significant loss of viability in dopaminergic neurons, although their selectivity varies, the least selective being PSI (Reaney et al. 2006).

Mesenchymal stem cells (MSCs) are present in adult bone marrow and represent < 0.01% of all nucleated bone marrow cells. MSCs are themselves capable of multipotency, with differentiation under appropriate conditions into chondrocytes, skeletal myocytes, and neurons (Makino et al. 1999; Pittenger et al. 1999; Woodbury et al. 2000). The application of MSCs in neurodegenerative diseases has seldom been studied. Mazzini et al. (2003) in a study of MSCs transplantation into spinal cord of patients with amyotrophic lateral sclerosis reported that most patients showed a slowing down of the linear decline or increasing muscle strength. Li et al. (2001) reported that using 1-methyl-4-phenyl-1,2,3,6-Tetrahydropyridine (MPTP)-treated PD model, MSCs injected intrastriatally survived, expressed dopaminergic protein tyrosine hydroxylase (TH) immunoreactivity, and promoted functional recovery. Very recently, we reported an open-label trial of hMSCs in patients with multiple system atrophy, demonstrating that hMSCs therapy delayed the progression of neurological deficits with achievement of functional improvement (Lee et al. 2007). Until now, however, there was no study of the application of MSCs in a setting similar to clinical PD with the viewpoint of neuroprotective strategies. The aim of this study was to investigate whether cell therapy with MSCs had a protective effect on progressive dopaminergic neuronal loss in vitro and in vivo, using MG-132. We also evaluated whether transplanted MSCs had the capacity to differentiate into TH-immunoreactive neurons and brought about functional recovery.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Result
  5. Discussion
  6. Acknowledgements
  7. Conflicts of Interest
  8. References
  9. Supporting Information

Antibodies

Antibodies and dilutions used in this study were included: mouse anti-TH (1 : 2000 dilution for brain tissue; 1 : 7500 dilution for cell culture, Pel-freez, Arkansas, USA), rabbit anti-TH (1 : 2000 dilution for brain tissue; 1 : 7500 dilution for cell culture, Chemicon, Temecula, CA, USA), mouse anti-nuclear matrix (NuMA, 1 : 100 dilution, Calbiochem, San Diego, CA, USA), rabbit anti-Synaptophysin (Syn, 1 : 200 dilution, Invitrogen, Carlsbad, CA, USA), OX-6 (1 : 200 dilution, BD Bioscience, Franklin Lakes, NJ, USA), CD34 (Immunotech, Marseille, France), and CD105 (SH-2) (Ancell, Bayport, MN, USA) for immunochemistry and immunofluorescence; CD34, CD45 (Immunotech), CD105 (SH-2) (Ancell), and anti-human CD73 (SH-3) (Alexis Biochemicals, San Diego, CA, USA) for Fluorescence-activated cell sorting (FACS) analysis; mouse anti-ubiquitin (Ub, 1 : 200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-3 (1 : 1000 dilution, Cell Signaling Tech., Beverly, MA, USA) for western blot. The different primary antibodies were co-detected by immunofluorescence, using goat anti-mouse IgG Alexa Fluor-488 (green) and goat anti-rabbit IgG Alexa Fluor-594 (red) (1 : 200, Molecular probes, Eugene, OR, USA).

Isolation of human MSCs (hMSCs) and rat MSCs (rMSCs)

Bone marrow aspirates (10 mL) were obtained from the iliac crests of human donors. The aspirates were mixed with 10 mL of phosphate-buffered saline (PBS). rMSCs were isolated from the bone marrow of adult Sprague-Dawley (SD) rats (8-week-old SD rats) as described (Liu et al. 2006). The mononuclear cell layer was isolated by Ficoll-Hypaque, washed in PBS, plated in polystyrene plastic 100 mm culture dishes, cultivated in low-glucose Dulbecco’s modified Eagles’ medium (Gibco-BRL, Grand Island, NY, USA), containing 10% fetal bovine serum (Hyclone, Irvine, CA, USA) and 1% penicillin/streptomycin (P/S, Sigma, St Louis, MO, USA), and maintained in a humidified incubator at 37°C under 5% CO2. When these primary cultures reached 80% confluence, the cells were harvested using 0.25% trypsin and subcultured. At passage 6, hMSCs were characterized by immunofluorescence and FACS analysis, and were injected into rats via tail vain.

Administration of proteasome Inhibitors and hMSCs

Male Sprague-Dawley (SD) rats (200–220 g) were used. The appropriate quantity of MG-132 was freshly prepared by dissolving in 50 μL dimethylsulfoxide. Before injection, this was then diluted to 0.5 mL with normal saline and injected subcutaneously (3 mg/kg) on alternate days (Monday, Wednesday, and Friday) over 2 weeks (six times in total). Control rats were injected with dimethylsulfoxide alone, using the same administration method. The injection of hMSCs (1 × 106) started 3 weeks after the last administration of MG-132 through tail vein. hMSCs injection was performed weekly for 3 weeks (3 × 106 cells in total). The experimental schedule is illustrated in Fig. 1. The injection of rat MSCs (1 × 106) was performed using same administration method. The animal work was approved by the respective Institutional Animal Care and Use Committees.

image

Figure 1.  Timeline of treatment and analysis. Before MG-132 administration, the rats were trained on the rotarod for 1 week. MG-132 (3 mg/kg) or vehicle (dimethylsulfoxide) as a control was injected subcutaneously 6 times over a period of 2 weeks. The injection of human mesenchymal stem cells (1 × 106) started 3 weeks after the last administration of MG-132 through the tail vein and performed weekly for 3 weeks. Rats were killed for immunohistochemistry, western blot, and RT-PCR at 8, 10, and 13 weeks after first MG-132 administration. B: behavior test.

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Primary culture of mesencephalic tissue and proteasome inhibitor treatment

Dopaminergic neurons were cultured from the SN of embryonic day 14 SD rats (Chung et al. 2001). The tissues were incubated with 0.01% trypsin in Ca2+−, Mg2+− free Hanks’ balanced salt solution (CMF-HBSS) for 10 min at 37°C. The cultures were rinsed twice in RF (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 6 mg/mL glucose, 204 μg/mL l-glutamine, and 100 U/mL P/S) to inhibit trypsin and were then dissociated into single cells by trituration. Dissociated cells were plated on 12-mm round aclar plastic coverslips or culture slides pre-coated with 0.1 mg/mL poly-d-lysin and 4 μg/mL laminin, and seeded in 12- and 24-well culture plates at a density of 2.0 × 106 cells/well and 1.0 × 105 cells/coverslip, respectively. The cells were incubated in a humidified incubator at 37°C, 5% CO2 for 24 h. At 2 days, the culture medium was replaced with chemically defined serum-free medium, composed of Ham’s nutrient mixture (F12-DMEM) with 1% ITS (insulin, transferrin, selenium), glucose, l-glutamine and P/S. At 4 days, the cultures were pre-treated with MG-132 (10 μM) for 2 h, and then primary cultures of mesencephalic tissue were co-cultured with 3 × 104 or 3 × 105 hMSCs/ well (using transwell) for 24 h. The co-culture cells were used for immunochemistry, western blot, and caspase-3 activity assay.

Tissue preparation

For immunohistochemistry, the animals were perfused with saline solution, containing 0.5% sodium nitrate and heparin (10 U/mL), and were fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB, ∼ 200 mL/rat). The brains were removed from the skulls, post-fixed overnight in buffered 4% paraformaldehyde at 4°C and stored in a 30% sucrose solution for 1–2 days at 4°C, until they sank. They were then sectioned to obtain a 30 μm coronal section or, after embedded in paraffin, 4 μm sections from the SN for immunofluorescence. The 30 μm coronal sections were stored in tissue stock solution (30% glycerol, 30% ethylene glycol, 30% 3 times distilled water, 10% 0.2 M PB) at 4°C until required. For western blotting and gas chromatography-mass spectrometry (GC-MS), animals were killed 13 weeks after first MG-132 treatment, and the SN and striatum were rapidly removed from the brains and frozen at −70°C.

FACS analysis

The characterization of hMSCs was evaluated by FACS analysis as previously described (Jankowski et al. 2003). Briefly, cell suspensions were washed twice with PBS containing 0.1% bovine serum albumin (BSA). For direct assays, 1 × 105 cells/mL were incubated with FITC-conjugated CD34, CD45 for negative marker, CD105 (SH-2), anti-human CD73 (SH-3) for positive marker at 4-C for 30 min, and then washed twice with PBS containing 0.1% BSA. The cells were analyzed by cytometric analysis using an EPICS XL flow cytometry (Becton-Dickinson, San Diego, CA, USA) with EXPO32 software.

Immunocytochemisrty and Immunohistochemistry

The 30 μm coronal brain sections and co-cultured cells were rinsed twice in PBS and incubated in 0.2% Triton X-100 for 30 min at room temperature (RT). They were rinsed three times with 0.5% BSA in 1 × PBS for blocking. After blocking, they were incubated overnight at RT or 4°C with primary antibodies (TH, NuMA, OX-6). After 24 h, primary cultures of mesencephalic tissue and brain sections were rinsed three times with 0.5% BSA in 1 × PBS (10 min/rinse) and incubated with the appropriate biotinylated secondary antibody and avidin–biotin complex (Elite Kit; Vector Laboratories, Burlingame, CA, USA) for 1 h at RT. Bound antibodies were visualized by incubating with 0.05% diaminobenzidine– HCl and 0.003% hydrogen peroxide in 0.1 M PB. The primary mesencephalic cultures and brain sections were rinsed with 0.1 M PB for diaminobenzidine inhibition. Immunostained cells were analyzed by bright-field microscopy.

Immunofluorescence double labeling

hMSCs, the 4 μm paraffin wax-embedded tissue sections, and the 30 μm coronal brain sections were rinsed twice in PBS and incubated in 0.2% Triton X-100 for 30 min at RT. They were rinsed three times with 0.5% BSA in 1 × PBS for blocking. After blocking, they were incubated overnight at RT or 4°C with primary antibodies (TH + NuMA, TH + syn, NuMA + syn, CD105, CD34). After 24 h, they were rinsed three times in 0.5% BSA in 1 × PBS (10 min/rinse), and incubated with goat anti-mouse IgG (Alexa Fluor-488, green) and goat anti-rabbit IgG (Alexa Fluor-594, red) secondary antibodies for 1 h at RT. hMSCs nuclei were counterstained with Hoechst 33342 (1 : 1000 dilution, Molecular Probes). hMSCs and brain tissues were then washed and mounted using Prolong Antifade Kit (Molecular Probes). Stained tissues were viewed using an Olympus I × 71 confocal laser scanning microscope (Olympus, Tokyo, Japan). To analyze the localization of different antigens in double-stained samples, immunofluorescence images were created from the same tissue section and merged using interactive software.

Western blot analysis

Brain tissues and co-cultured cells were dissolved in ice-cold lysis buffer (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2, 1 mM dithiothretol, 0.1 mM phenylmethylsulfonyl fluoride plus protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The lysates were centrifuged (20 min, 14 000 g, 4°C) and supernatants were transferred to fresh tubes. Proteins were analyzed using the Bio-Rad Protein Assay Kit (Hercules, CA, USA). Equal amounts of protein (50 μg) were loaded in each lane with loading buffer containing 0.125 M Tris–HCl, pH 6.8, 20% glycerol, 4% sodium dodecyl sulfate, 10% mercaptoethanol, and 0.002% bromophenol blue. Samples were boiled for 5 min before gel loading. Proteins so separated were transferred electrophoretically to polyvinylidiene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were washed in Tris-buffered saline solution with 2.5 mM EDTA (TNE) and then blocked in TNE containing 5% skim milk for 1 h. Membranes were incubated overnight at 4°C with primary antibodies specific for Ub, caspase-3 and actin (1 : 2000; Santa Cruz). After washing, the membranes were incubated with secondary antibodies (1 : 2000; Amersham, Piscataway, NJ, USA) for 1 h at RT, and washed again. The blots were finally developed with the ECL western blotting detection reagents (Amersham). For semiquantitative analysis, the density of immunoblot bands was measured with the computer imaging (Fugi film).

Caspase-3 activity assay

Caspase-3 activity was measured using the Caspase-3 Colorimetric activity assay system (Chemicon, USA & Canada). Co-culture cells were washed with ice-cold PBS and caspase-3 activity assay was then performed according to manufacturer’s instructions.

GC-MS to assess dopamine levels

Tissue homogenates of striatum were centrifuged (20 min, 14 000 g, 4°C) and the supernatant was transferred to a fresh tube for the dopamine assay using GC-MS. GC–MS analyses in both scan and selected ion monitoring modes were performed using an Agilent 6890 gas chromatograph, interfaced to an Agilent 5973 mass-selective detector (70 eV, electron impact mode) and installed with an Ultra-2 (5% phenyl–95% methylpolysiloxane bonded phase; 25 m×0.20 mm i.d., 0.11 μm film thickness) cross-linked capillary column (Agilent Technologies, Atlanta, GA, USA). The temperatures of the injector, interface, and ion source were 260, 300, and 230°C, respectively. Helium was used as carrier gas at a flow rate of 0.5–1 mL/min in a constant flow mode. Samples were introduced in split-injection mode (10 : 1) and the oven temperature was initially at 170°C for 2 min, and programmed to increase at 5°C/min to 205°C and at 2°C/min to 220°C and finally to 30°C/min to 300°C. The mass range scanned was 50–800 U at a rate of 0.42 scan/s. All GC–SIM–MS runs were performed in triplicate.

Adhesive-removal test

All rats were familiarized with the testing environment. In the initial test, two small pieces of adhesive-backed paper dots (of equal size, 113.1 mm2) were used as bilateral tactile stimuli occupying the distal-radial region on the wrist of each forelimb. The rat was then returned to its cage. The time to remove each stimulus from forelimbs was recorded on trials per days. Before MG-132 administration, the animals were trained for 2 days. All rats (n = 8/group) were evaluated by removal time of dots (Hernandez and Schallert 1988).

Stereological cell counts

Unbiased stereological estimations of the total number of the staining cells in the SN were made using an optical fractionator, as previously described with some modifications (Kirik et al. 1998). This sampling technique is not affected by tissue volume changes and does not require reference volume determinations (West et al. 1991). The sections used for counting covered the entire SN, from the rostral tip of the pars compacta back to the caudal end of the pars reticulate. This generally yielded 8–9 sections in a series. Sampling was performed using the Olympus C.A.S.T.-Grid system (Olympus Denmark A/S, Denmark), using an Olympus BX51 microscope, connected to the stage and feeding the computer with the distance information in the z-axis. The SN was delineated at 1.25 × objective. A counting frame (60%, 35, 650 μm2) was placed randomly on the first counting area and systematically moved though all counting areas until the entire delineated area was sampled. Actual counting was performed using a 40 × oil objective. Guard volumes (4 μm from the top and 4–6 μm from the bottom of the section) were excluded from both surfaces to avoid the problem of lost cap, and only the profiles that came into focus within the counting volume (with a depth of 10 μm) were counted. The total number of stained cells was calculated according to the optical fractionator formula (West et al. 1991).

Statistical analysis

The Mann–Whitney test was used to compare the means in pairs of groups. p-values < 0.05 were considered statistically significant. Statistical analyses were performed using commercially available software (version 10.0; spss Inc., Chicago, IL, USA).

Result

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Result
  5. Discussion
  6. Acknowledgements
  7. Conflicts of Interest
  8. References
  9. Supporting Information

Characterization of hMSCs

FACS analysis demonstrated that hMSCs expressed CD105 and CD73, positive markers for hMSCs, and did not express CD 45 and CD 34, negative markers for hMSCs (Fig. 2a). Immunofluorescent labeling showed that hMSCs were positive for CD 105 and negative for CD 34 (Fig. 2b).

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Figure 2.  Flow cytometric analysis (a) and immunofluorescent labeling of human mesenchymal stem cells (b). Scale bar: 100 μm.

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Effect of hMSC on dopaminergic neurons in vitro

After 2 h of treatment with a proteasome inhibitor (MG-132, 10 μM), dopaminergic neurons in primary mesencephalic cultures were treated with hMSCs for 24 h. Immunocytochemical analyses revealed that hMSC treatment increased dopaminergic neuronal survival in MG-132 treated cells (Fig. 3a). Quantitative analysis of TH+ cells showed that hMSC treatment significantly decreased dopaminergic neuronal loss induced by MG-132 (p < 0.01, Fig. 3b). In addition, hMSCs treatment resulted in a significant reduction in the cleaved form of caspase-3 (Fig. 3c) and caspase-3 activity (Fig. 3d).

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Figure 3.  Effects of human mesenchymal stem cells on dopaminergic neurons in vitro. Primary cultures of mesencephalic tissue were pretreated with MG-132 for 2 h and then co-cultured with hMSCs for 24 h. In the hMSCs co-culture group, there was markedly decreased MG-132-induced TH-immunopositive cell death on immunocytochemistry (a) and quantitative analysis of TH-immunopositive cells (n = 3, b). hMSCs treatment resulted in a significant reduction in the cleaved form of caspase-3 (c) and caspase-3 activity (n = 3, d) following MG-132 treatment. Scale bar: 100 μm. *p < 0.01, **p < 0.001.

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Progressive decline in the number of TH-immunoreactive cells in MG-132-treated rats

Rats that received systemic injection of MG-132 did not show evidence of systemic toxicity, and none died. MG-132 did not cause significant weight change, compared with controls. At 2 and 4 weeks after the initiation of treatment, mean body weights were 291.8 ± 5.6 g and 381 ± 10 g, respectively, for controls (n = 5), compared with 290.8 ± 5.1 g and 386 ± 9.4 g, respectively, for MG-132-treated rats (n = 8).

Immunohistochemical analysis showed that there was a progressive decline in the number of TH-immunoreactive cells in the SN of MG-132-treated animals (Fig. 4a). TH-immunoreactive neuronal loss was more prominent in the lateral than the medial region of the SN. Quantification of the extent of neuronal loss assessed by stereological analysis revealed that TH-immunoreactive cell loss was 23 ± 7.2% (p < 0.05, n = 5 per group) at 8 weeks, 70 ± 9.5 at 10 weeks (p < 0.01, n = 5 per group), and 92 ± 7.2 at 13 weeks (p <0.001, n = 5 per group; Fig. 4b).

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Figure 4.  Progressive decline in the number of TH-immunoreactive cells in MG-132-treated rats. Immunohistochemical analysis showed that there was a progressive decline in the number of TH-immunopositive cells in the SN of MG-132-treated animals (a). Quantification of the extent of neuronal loss assessed by stereological analysis revealed that TH-immunopositive cell loss was 23 ± 7.2% at 8 weeks, 70 ± 9.5% at 10 weeks, and 92 ± 7.2% at 13 weeks (n = 5, b). Scale bar: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

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Effect of cell therapy with hMSC on animals treated with MG-132

Brain tissue was harvested at 8 weeks after the first hMSCs injection for immunohistochemical, western blot, and GC-MS analysis. Immunohistochemical analysis showed that hMSCs treatment dramatically reduced the decline in the number of TH-immunoreactive cells in the SN of MG-132-treated rats (Fig. 5a). Stereological analysis revealed that the number of TH-immunoreactive cells was significantly higher in hMSCs treatment group than in the group treated with MG-132 alone (p < 0.05), showing about a 50% increase in the survival of TH-immunoreactive cells in the SN (Fig. 5b). Consistent with the loss of dopaminergic neurons in the SN, the dopamine level in the striatum was significantly decreased in MG-132-treated rats compared with controls (p < 0.01). However, hMSCs treatment significantly increased the dopamine level in the striatum of MG-132-treated rats (p < 0.05, Fig. 5c), consistent with increased survival of TH-immunoreactive cells in the SN after hMSCs treatment.

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Figure 5.  Effect of cell therapy with human mesenchymal stem cells (hMSCs) on animals treated with MG-132. Immunohistochemical analysis showed that hMSCs treatment dramatically reduced the decline in the number of TH-immunopositive cells in the SN of MG-132-treated rats (a). Stereological analysis revealed that the number of TH- immunopositive cells was significantly higher in hMSCs treatment group than in the group treated with MG-132 alone (n = 5; p < 0.05, b). Dopamine levels in the striatum, assessed by gas chromatography-mass spectrometry, were significantly decreased in MG-132-treated rats, compared with controls (p < 0.01); however, hMSCs treatment significantly increased the dopamine level in the striatum of MG-132-treated rats (n = 5; p < 0.05, c). MG-132 treatment resulted in accumulation of poly-ubiquitinated proteins and markedly increased OX-6 immunoreactivity; however, hMSCs treatment markedly decreased the accumulation of poly-ubiquitinated proteins and OX-6 immunoreactivity in MG-132-treated rats (d and e). In rats treated with hMSCs, there was a significant reduction in the cleaved form of caspase-3 (f), compared with MG-132 treated rats (n = 3, g). Scale bar: 100 μm. *p < 0.05, ***p < 0.01.

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Since inhibition of proteasomal activity increases the level of ubiquitinated proteins as a consequence of reduced clearance of proteins by the UPS (Rideout and Stefanis 2002), we evaluated the level of high molecular weight poly-ubiquitinated proteins in MG-132-treated animals. MG-132 treatment resulted in accumulation of poly-ubiquitinated proteins; however, hMSCs treatment markedly decreased this accumulation of poly-ubiquitinated proteins in MG-132-treated rats (Fig. 5d). To determine whether hMSCs treatment modulated microglial activation, the SN was immunostained with OX-6, a marker for activated microglia. There was a marked increase in OX-6 immunoreactivity in MG-132-treated rats; however, hMSCs treatment in MG-132-treated rats markedly decreased OX-6 immunoreactivity (Fig. 5e). In rats treated with hMSCs, there was a marked reduction in the cleaved form of caspase-3 compared with MG-132-treated rats (Fig. 5f and g).

To eliminate the possibility of immune enhancement form a xenograft model, we reanalyzed the effect of MSCs on modulation of microglial activation and survival of TH-immunoreactive cells using rat MSCs. As in hMSCs, rat MSCs treatment significantly decreased microglial activation and TH-immunoreactive cell loss compared with the group treated with MG-132 alone (Supporting information Fig. S1).

Histological analysis of transplanted hMSCs in animal model of PD induced by MG-132

To determine whether transplanted hMSCs would survive, we identified hMSCs in the SN of MG-132-treated rats using human-specific NuMA staining. In controls and in animals treated with MG-132 alone, there were no NuMA-immunoreactive cells (Fig. 6a). In contrast, NuMA-immunoreactive cells were observed in animals treated with hMSCs. The number of NuMA-immunoreactive cells was 51957 ± 35, which corresponded to about 1.7% of the total injected hMSCs (Fig. 6b). To determine whether the transplanted hMSC possessed the phenotype of TH neurons, we co-stained NuMA-immunoreactive cells with antibodies to TH (Fig. 6c). The number of cells double-stained with NuMA and TH was 687 ± 35, about 36% of the NuMA-immunoreactive cells (Fig. 6d). Furthermore, we evaluated whether the cells positive for NuMA and TH staining were also immunoreactive for human-specific synaptophysin, using the thin (4 μm) paraffin sections. As shown in Fig. 5e, transplanted hMSC was triple-stained with NuMA, TH, and synaptophysin, indicating that the transplanted hMSC harbored phenotype of TH as well as human-specific synaptophysin.

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Figure 6.  Histological analysis of transplanted human mesenchymal stem cells (hMSCs) in MG-132-treated rats. The existence of hMSCs in the substantia nigra (SN) of MG-132 treated rats was identified by human specific NuMA staining. There were no NuMA-immunoreactive cells in controls or MG-132-treated rats (a). However, NuMA-immunoreactive cells were observed in animals treated with hMSCs, the number being about 1.7% of the total injected hMSCs (n = 5, b). About 35.7% of NuMA-immunoreactive cells possessed the phenotype of TH neurons (n = 5; c, d). Using thin (4 μm) paraffin wax sections, transplanted hMSC was triple-stained with NuMA, TH, and synaptophysin, indicating that transplanted hMSC exhibited the TH phenotype and expressed human-specific synaptophysin (e, i; NuMA (green), TH (red), ii; NuMA (green), Syn (red), iii; TH (green), Syn (red)). Scale bar: 20 μm (red), 100 μm (black).

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Behavioral assessment

The mean time to remove each stimulus from forelimbs was significantly higher in MG-132-treasted rats than control rat throughout study period (p < 0.05). Compared with MG-132-treated rats, hMSCs administration in MG-132-treated rats had a tendency of decrease in mean removal time and this tendency was statistically significant at 3, 4, 6, 7, 10 weeks after MG-132 treatment (p < 0.05, Fig. 7).

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Figure 7.  Adhesive-removal test. The mean time to remove each stimulus from forelimbs was significantly higher in MG-132-treasted rats than control rat throughout study period (p < 0.05). Compared with MG-132-treated rats, hMSCs administration in MG-132-treated rats had a tendency of decrease in mean removal time and this tendency was statistically significant at 3, 4, 6, 7, 10 weeks after MG-132 treatment (p < 0.05). *p < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Result
  5. Discussion
  6. Acknowledgements
  7. Conflicts of Interest
  8. References
  9. Supporting Information

Although PD is the only chronic neurodegenerative disease for which there are effective for symptomatic treatment of dopaminergic regimens, these therapies do not change the progressive nature of PD and moreover, some axial symptoms of gait-freezing and postural instability, which give more disability than tremor and rigidity, do not respond to dopaminergic therapies. In addition, as PD progress, dopaminergic therapies results in disabling drug-induced motor complications, such as wearing off and dyskinesia, and a variety of non-motor symptoms, such as dementia, autonomic dysfunction, and mood disorders also emerge, which is comparatively disabling as much as motor symptoms (Adler 2005). Therefore, neuroprotective strategy that favorably influences the disease process or underlying pathogenesis to produce enduring benefits for PD patients is required.

Many candidates for neuroprotective agents have been tested in animal models of PD. However, most drug candidates have been tested in acute or subacute neurotoxin-induced animal models and the compounds are administered before the development of PD in the model by neurotoxin. To truly test neuroprotective agents in animal models of PD similar to clinical setting, an animal model of PD should have the same chronic, progressive nature and administration of candidate drugs should start after neuronal loss in the SN has started (Emborg 2004). After introduction of chronic progressive PD animal model using systemic injection of proteasome inhibitor (McNaught et al. 2004), there has been extensive debate concerning the outcome of the nigral neuronal loss; some groups demonstrated reproducible PD-like neuropathology and decrease in locomotor activity (Schapira et al. 2006; Zeng et al. 2006), whereas other groups failed to induce a reproducible model of PD (Bove et al. 2006; Kordower et al. 2006; Manning-Bog et al. 2006). Regarding the discrepancy, MaNaught and Olanow stated that several unidentified factors, such as variation in the properties of proteasome inhibitor, environmental factors, and difference in dosing and brain bioavailability of the toxin may account for this poor reproducibility. In this study, chronic administration of MG-132 in this study clearly brought about a progressive decline in the number of TH-immunoreactive cells (∼ 23% TH-immunoreactive cell loss at 8 weeks to ∼92% TH-immunoreactive cell loss at 13 weeks), similar to the progressive PD model conducted by McNaught et al. (2004). This decline in TH-immunoreactive neurons in the SN was accompanied by the decreased dopamine levels in the striatum as well as mean removal times on adhesive-removal test, compared with controls. Regarding the treatment time of candidate drugs, treatment of hMSCs in this study began 2 h after MG-132 administration in mesencephalic cultures and 3 weeks after initial administration of MG-132 in animals, respectively.

The present study demonstrated that hMSC treatment significantly decreased dopaminergic neuronal loss resulting from MG-132 treatment both in vitro and in vivo, showing a 50% increase in the survival of TH-immunoreactive cells in the SN. Increased dopamine level in the striatum and a tendency for reduction in behavioral deterioration after hMSCs treatment were consistent with increased survival of dopaminergic neurons following MSCs treatments in MG-132-treated animals. The mechanism of neuroprotective effects in hMSCs seems to be more complex and pleiotrophic, which may be mediated by trophic mechanisms. First, this study demonstrated that hMSCs treatment markedly decreased accumulation of poly-ubiquitinated proteins and caspase-3 activity in vitro as well as in vivo following MG-132 treatment. Second, hMSC treatment had anti-inflammatory effect, showing significantly reduced microglial activation in MG-132-treated animals. Although initially triggering events in dopaminergic neuronal death in PD remains unknown, apoptosis and altered proteasome activity play pivotal roles in the pathogenesis of PD. DNA fragmentation and chromatin clumping in the dopaminergic neurons, coupled with up-regulation of signals associated with apoptosis in PD patients supports that dopaminergic neurons would be dead by apoptosis in PD (Tatton et al. 2003). Recent genetic, postmortem, and experimental studies have suggested that proteasomal dysfunction might play an important role in the accumulation of toxic proteins and, consequently, neurodegeneration in the SN, mediating by an imbalance between degradation and clearance of abnormal proteins (Olanow and McNaught 2006). Evidence has been presented in several studies of in vitro or in vivo disease models that inhibition of the inflammatory response can prevent degeneration of nigrostriatal dopaminergic neurons. For example, sodium salicylate, cyclooxygenase-2 inhibitor, or minocycline have been shown to significantly reduce striatal dopaminergic depletion and dopaminergic neuronal loss induced by MPTP or LPS-models (Aubin et al. 1998; Du et al. 2001; He et al. 2001; Sairam et al. 2003; Teismann et al. 2003). Furthermore, a recent epidemiological study has demonstrated that regular use of nonsteroidal anti-inflammatory drugs was associated with a 45% lower risk of PD compared with a nonuser (Chen et al. 2003a), supporting the neuroprotective effects of nonsteroidal anti-inflammatory drugs in the development or progression of PD. It is known that the MSCs can be mobilized from the marrow and produce a variety of trophic factors that inhibit apoptosis and inflammation (Caplan and Dennis 2006).

MSCs characteristically migrate towards damaged tissues in an animal model of ischemia as well as PD model using 6-hydroxydopamine, possibly in response to signals that are up-regulated under injury condition (Li et al. 2001; Chen et al. 2003b; Hellmann et al. 2006). Although signals that guide MSCs to damaged brain are as yet unknown, chemokines released from damaged brain and their receptors may be involved. Of those, recent studies have demonstrated that stromal cell-derived factor-1 (SDF-1α) and its receptor CXCR4 play an important role in homing of MSCs to ischemic brain lesions (Stumm et al. 2002; Chamberlain et al. 2007). SDF-1α is widely expressed in the brain including cortex, cerebellum, and globus pallidus as well as the SN pars compacta (Banisadr et al. 2003). Therefore, as in ischemic brain lesions, it is speculated that damage in SN caused by MG-132 may increase the expression of SDF-1α and CXCR4, leading to recruitment of MSCs to substantial nigra. In this study, the number of surviving hMSCs in the SN 6 weeks after last hMSCs administration was approximately 1.7% of total injected hMSCs. We believe that these migrated cells may contribute to the production of trophic factors and inhibit microenvironmental cascade of the neurodegenerative process in nigral dopaminergic neurons.

Recent studies indicate that human MSCs can be induced to differentiate into neuron-like cells (Makino et al. 1999; Pittenger et al. 1999; Woodbury et al. 2000; Mareschi et al. 2006). Moreover, Blondheim et al. (2006) demonstrated that MSCs had an expression of several specific neuronal markers and transcriptional factors, of which a large proportion of the genes was participating in the neuro-dopaminergic system, and Barzilay et al. (2008) recently reported a serum-free controlled differentiation protocol that would yield dopamine-producing cells from MSCs, with more than 30% of the cells expressing significant levels of TH. There have been a few reports that MSCs can differentiate into TH-immunoreactive neurons in vivo study but the results are contradictory. Li et al. (2001) reported that MSCs injected intrastriatally exhibited the phenotype of dopaminergic neurons in MPTP animal model, and Blondheim et al. (2006) and Offen et al. (2007) demonstrated that intrastriatal transplantation of undifferentiated and differentiated MSCs in 6-hydroxydpomine animal model led to express TH in striatum. However, Ye et al. (2007) did not find BrdU and TH-immunoreactive cell in the striatum and suggested that functional recovery in MSCs-treated rat may not be associated with differentiation of MSCs into TH-immunoreactive neurons. Our data demonstrated that approximately 35.7% of surviving hMSCs in the SN displayed TH-positive immunoreactivity and, furthermore, TH-immunoreactive cells could be immunostained with human-specific synaptophysin, suggesting that TH-immunoreactive cells may have a dopaminergic function. Taken with the trophic effects of anti-apoptosis, anti-inflammation, and decreased poly-ubiquitinated proteins, differentiation of hMSCs into functional TH-immunoreactive neurons may partly contribute to functional recovery and decreased neurodegeneration in MG-132-treated animals.

In addition to the molecular and cellular benefits of hMSCs, cell therapy with hMSCs has an advantage in clinical applications. hMSCs can be easily harvested from self bone marrow, cultured in vitro, and administered to patients via various roots including intravenous, intraarterial, intrathecal, or intralesional infusion. In contrast with embryonic stem cell therapy, there is no immunological rejection, and cell therapy with hMSCs is free from ethical issues. Importantly, regarding the safety of hMSCs application, our group reported recently that cell therapy with hMSCs in patients with multiple system atrophy and ischemic stroke was feasible and safe (Bang et al. 2005; Lee et al. 2007). This is the first report of hMSCS application in a setting similar to clinical PD with the viewpoint of neuroprotective strategies, and our data postulate that hMSCs, as a possible neuroprotective strategy in PD treatment, may be applicable clinically.

In conclusion, we have shown that hMSC treatment has a protective effect against dopaminergic neuronal death induced by MG-132 in vitro and in vivo. Complex mechanisms mediated by trophic effects of hMSCs and differentiation of hMSCs into functional TH-immunoreactive neurons may work in the neuroprotective process. In addition, neuroprotective strategy using hMSCs may be applicable clinically.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Result
  5. Discussion
  6. Acknowledgements
  7. Conflicts of Interest
  8. References
  9. Supporting Information

This research was supported by a grant from Stem Cell Research Center of the 21st Century Frontier Research Program (SC-4111) funded by the Ministry of Science and Technology, Republic of Korea.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Result
  5. Discussion
  6. Acknowledgements
  7. Conflicts of Interest
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Result
  5. Discussion
  6. Acknowledgements
  7. Conflicts of Interest
  8. References
  9. Supporting Information

Fig. S1 The injection of rat MSCs (rMSCs, 1 × 106) started 2 weeks after the last administration of MG-132 through tail vein. rMSCs injection was performed every 3 days for 1 week (2 × 106 cells in total) and brain tissue was harvested at 2 weeks after the first rMSCs injection. Immunohistochemical analysis showed that rMSCs treatment dramatically decreased the loss of TH- immunopositive cells in the SN of MG-132-treated rats and OX-6 stained microglial activation (a). Stereological analysis revealed that the number of TH-immunopositive cells was significantly higher in rMSCs treatment group than in the group treated with MG-132 alone (b, n = 5)). Scale bar: 100 μm. *p ≤ 0.05, **p ≤ 0.01

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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.