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TISSUE-SPECIFIC STEM CELLS |
aDepartment of Biomedical Sciences, Iowa State University, Ames, Iowa, USA;
bDepartment of Neurology, University of Cincinnati, Cincinnati, Ohio, USA
Key Words. 3-Nitropropionic acid • Growth factor • Huntington's disease • Neuroprotection • Regeneration • Striatum Superoxide dismutase • Transplantation
Correspondence: Correspondence: Jitka Ourednik, Ph.D., Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA, Telephone: +1-515-294-6449; e-mail: joured{at}iastate.edu
Received on March 27, 2007;
accepted for publication on October 8, 2007.
First published online in STEM CELLS EXPRESS October 25, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgmentsReferences |
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To be able to realize their therapeutic purpose, grafted and endogenous NSPs have to resist the hostile pathological microenvironment as much as or even better than the other host cell populations. This is, indeed, reflected in the increased "vigilance" of their antioxidant defense mechanisms in presence of oxidative stress, as we demonstrated in our recent article in this journal [8].
The aim of the present study was to examine the dynamics of the dialog between NSPs and their cellular environment upon exposure to 3-NP, an inhibitor of the mitochondrial respiratory complex II [9]. Such treatment leads to deficits in cellular energy metabolism such as depletion in ATP levels and formation of excess amounts of reactive oxygen and nitrogen species [10]. The resulting effects replicate most of the clinical and pathological features of Huntington's disease (HD) and include excitotoxicity and oxidative stress, both of which are likely to be major causes of apoptosis and neuronal cell death [10]. The central questions of our study were: (1) can NSPs, if grafted into adult brain or added to the culture of primary neural cells (PCs) prior to the insult, enhance 3-NP resistance, and (2) what are the involved intercellular signaling events related to oxidative stress that lead to the increased 3-NP resistance.
In vivo, we observed that adult mice grafted with NSPs prior to their treatment with 3-NP exhibit reduced behavioral symptoms and less severe damage of striatal cytoarchitecture caused by oxidative stress than sham-transplanted controls. First histological data obtained from these experiments suggested that this amelioration correlates with neuroprotective effects and distribution patterns of donor cells in the host brain. Subsequently, in an in vitro approach allowing better control and reproducibility of the experimental conditions and molecular analyses, we focused on changes in the cellular expression of growth factors (GFs) such as ciliary neurotrophic factor (CNTF) and vascular endothelial growth factor (VEGF), and on correlation between their presence and the production of detoxifiers of reactive oxygen species (ROS). A presence of exogenous NSPs before the 3-NP treatment was chosen in order to favor protection of surrounding elements from the arriving toxin rather than their rescue and/or regeneration. From a therapeutic point of view, such "proactive" neurotransplantation could become useful in, for example, Huntington's disease or Ataxia Telangiectasia, where the onset of symptomatic disease manifestations is predetermined, or in other hereditary neurological disorders, for which new diagnostic tools might in future allow an early assessment of disease risk and approximate onset time.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgmentsReferences |
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Animals. Sixteen to 20 week old male C57BL/6 mice were housed and maintained at Iowa State University and all animal procedures carried out were approved by the Iowa State University Committee on Animal Care and adhered to NIH guidelines (Public Health Service Policy on Humane Care and Use of Laboratory Animals, 2002). The following experimental groups were used: (1) mice grafted with vehicle (Hank's balanced salt solution (HBSS)) and 3-NP injected: short term group, n = 12, long-term group, n = 10; (2) mice grafted with NSPs and 3-NP injected: short term group, n = 9, long-term group, n = 8; (3) mice grafted with 3T3 cells and 3-NP injected: short term group, n = 6, long-term group, n = 5; (4) intact mice: short term group, n = 6, long-term group, n = 5; (5) intact mice grafted with NSPs: short term group, n = 8, long-term group, n = 7. Mice from (1) and (3) were collectively defined as "mock-grafted", since both groups presented similar behavior and histology throughout this study. The mice were sacrificed at 3 days or 3 weeks post-3-NP treatment.
Transplantation. Grafting occurred unilaterally into the intact right striatum of behaviorally trained mice (see below). Anesthetized mice [0.5 mg of pentobarbital (Nembutal) per kg.b.w] received NSPs, 3T3 cells (both in 1.5 µl of HBBS, 75,000 cells/µl), or an equal volume of vehicle at the following stereotactic coordinates: 2 mm anterior to bregma, 1.5 mm lateral to the midline, and 2 mm along the dorso-ventral axis (see [6] for more details).
3-NP Treatment. One week after surgery, animals were subject to subacute striatal damage by 3-NP obtained by modification of the strategy employed by Fernagut et al. [11]. Briefly, the mice received systemic injections of the neurotoxin twice a day, 12 hours apart, for 6 days. The number of injections and the dosing were as follows: 4 x 20 mg/kg b. w. injections followed by 8 x 40 mg/kg b.w. injections, resulting in a total dose of 400 mg/kg b.w. Behavioral testing of the mice started with the first 3-NP injection (see experimental timeline in the supplementary online data).
Behavioral Training and Tests. All mice were trained daily in pole and string tests for 2 weeks prior to surgery following established protocols [11, 12] (see supplementary online data for more details).
Monitoring of the Grafted NSPs. Cells were labeled with enhanced green fluorescent protein (EGFP) using retroviral transduction leading to strong and stable green fluorescent protein expression. They were monitored by direct fluorescence or by immuno-detection of GFP.
Histological Analyses. Mice were sacrificed either 3 days or 3 weeks after the last 3-NP injection. Brains were removed, fixed in 4% paraformaldehyde in 0.1 M PBS, and processed for routine cryostat or polyester wax sectioning [6]. Serial, coronal, 20-µm thick sections were collected from both striata between coronal levels 151 and 232 as defined in [13] and stained with hematoxylin where required.
Immunohistochemistry. Rehydrated sections were blocked and immunostained over night at 4°C with Abs against calbindin (Calb; 1:500), neuronal nuclear antigen (NeuN; 1:300), 4-hydroxy-2-nonenal (HNE; 1:500) or SOD2 (1:200). Primary Abs were detected in a 2 hour incubation at RT with secondary Abs (1:500) coupled to the fluorochromes Alexa Fluor 488, 594, or 647 for immunofluorescence. Alternatively, 3,3' diaminobenzidine (DAB)-staining with biotinylated secondaries and the ABC peroxidase kit (Vector, Burlingame, CA, http://www.vectorlabs.com) was performed. Stains omitting primary Abs or using non-specific secondary Abs served as controls.
Cell Culture and In Vitro Assays. (See also supplementary online material for details.)
Neural Stem/Progenitor Cells (NSPs). NSPs isolated from the subventricular zone of newborn C57BL/6 mice and labeled with GFP (kindly provided by Dr. Kraus, Oslo, Norway) were used. The cells were grown, under standard serum-free conditions in Neurobasal (NB) medium 50 units/ml penicillin and 50 µg/ml streptomycin, heparin (8 µg/ml), basic fibroblast growth factor (bFGF; 20 ng/ml), and epidermal growth factor (EGF; 20 ng/ml), but using an antioxidant free B27 supplement by Invitrogen (catalogue No. 10,889-038). The cells underwent 2 passages in culture before their utilization.
Primary Neural Cells (PCs). For each set of assays, PCs derived from striatal tissue of 17 day old (E17) C57BL/6J mouse embryos were resuspended as heterogeneous cell population into poly-L-lysine-coated 24–well plates at 1 x 105 cells/cm2 in NB medium supplemented with B27, 10% horse serum, 500 µM L-glutamine, 25 µM L-glutamate, 200 units/ml penicillin, and 200 µg/ml streptomycin. These parallel cultures were incubated at 37°C as primary neuron-glia (PNGs) cultures in a humidified incubator under standard conditions. After 6 days (the time when cocultures with NSPs were started, see below), these cultures were composed of about 60% neurons and 40% glial cells. To obtain primary neuronal cultures (PNs), 24 hours after plating, the medium was changed to serum-free NB medium supplemented with B27, 500 µM L-glutamine, 200 units/ml penicillin, and 200 µg/streptomycin and treated on day 4 with 10 µM cytosine 1-β-D-arabinofuranoside for 24 hours to inhibit glial growth. Primary glial cell cultures (PGs) were grown as described previously [14].
NIH 3T3 Fibroblasts. Cells were grown in medium containing Dulbecco's Minimal Essential Medium supplemented with 10% FBS, L-glutamine (2 mM), and 50 units/ml penicillin and 50 µg/ml streptomycin.
Cocultures of NSPs with PCs. NSPs were added at 5 x 103 cells/well either directly to 6 day old cultures of PCs (PNGs, PNs, or PGs; 105 cells/well) or cocultured on a membranous polyester insert (6.5 mm diameter and 0.4 µm pore size) (Costar Transwell;Corning, Corning, NY, http://www.corning.com) above the primary cells. The coculture medium consisted of NB medium supplemented with B27, 500 µM L-glutamine, 200 units/ml penicillin, and 200 µg/ml streptomycin, heparin (8 µg/ml), bFGF (20 ng/ml) and EGF (20 ng/ml). PCs were also grown alone in the same medium, in order to assess possible influences of its composition on their behavior. None could be detected.
Cell Culture Paradigms. For each set of experimental assays, the following culture paradigms were used, with at least 3 independent parallel cultures per assay: (1) PCs only (no 3-NP), (2) NSPs only (no 3-NP), (3) PCs + 3-NP, (4) NSPs + 3-NP, (5) coculture of NSPs with PCs before the addition of 3-NP, (6) coculture of NSPs with PCs (no 3-NP), (7) coculture of 3T3 cells with PCs before the addition of 3-NP, and (8) coculture of 3T3 cells with PCs (no-3-NP). Cocultures of NSPs with PCs occurred either with or without separating polyester inserts (see above for specification).
Induction of Oxidative Stress In Vitro. Oxidative stress was induced in 7 day old PCs by treatment of PNGs with 0.1 mM 3-NP, PGs with 0.25 mM, and PNs with 0.05 mM 3-NP for 48 hours according to our previously established dose/time-response curves (see supplementary online material for more details).
Inhibition of GF Signaling. Neutralizing antibodies against VEGF, CNTF, and brain-derived neurotrophic factor (BDNF) were added to the cocultures 24 hours prior to the addition of 3-NP, at concentrations of 1, 2, and 0.5 µg/ml respectively. Their presence was maintained also after 3-NP was removed. In simultaneously processed 3-NP free controls, the addition of the neutralizing antibodies did not produce any toxic effects (not shown).
ATP Assay. ATP activity was assayed using the ATPlite Luminescence ATP detection system (Perkin Elmer, Boston, http://www.perkinelmer.com) in accordance with the manufacturer's directions. Luminescence was read on a Turner Designs microplate luminometer (Turner Designs, Sunnyvale, CA, http://www.turnerdesigns.com). Standardized calculations were expressed as percentage of ATP levels measured in vehicle treated wells.
Mitochondrial Activity and ROS Levels. Standard colorimetric 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) tests and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) fluorescence assays were performed on cell cultures as described previously in [8].
Detection of Apoptosis. Apoptotic cells in vitro were detected in a standard nuclear stain with the Hoechst dye 33342 and cells with apoptotic (condensed and/or fragmented) nuclei were quantified as described [15].
Immunocytochemistry. The following antibody specificities were used for cell characterization: nestin (Ab dilution 1:500) and Ki-67 (1:500) for detection of NSPs and proliferating cells; class III-β-tubulin (Tuj1; 1:300) and microtubule associated protein 2ab (MAP2ab; 1:500) as neuronal markers; glial fibrillary acidic protein (GFAP; 1:1000) and S-100β (1:1000) as astroglial markers; 2'3'-cyclic-nucleotide 3'-phosphodiesterase (CNP; 1:500) as marker for oligodendrocytes; monitored was also expression of CNTF (1:250), BDNF (1: 1,000), VEGF (1:250), GDNF (1: 500), NT-3 (1:500), and of the antioxidant SOD2 (1:500). Cells were processed as described [8] and stains analyzed using a Zeiss Axioplan (Carl Zeiss, Inc., Oberkochen, Germany, http://www.zeiss.com) two microscope. Images were captured with a Zeiss AxioCam digital camera and Axiovision 4 software.
Protein Quantification by ELISA. Contents in GFs were determined from collected culture media using an R & D Quantikine ELISA Immunoassay kit (R&D Systems, Minneapolis, MN, http://www.rndsystems.com) for VEGF and CNTF, and Promega Emax Immunoassay system (Promega, Madison, WI, http://www.promega.com) for BDNF following the recommendations of the manufacturer. Pure GF proteins provided in the kit were used to establish standard curves for quantification.
Western Blot Analysis. Forty-eight hours after 3-NP treatment, electrophoresis and electroblotting of cell lysates were performed as described [8]. The presence of SOD2 was revealed by incubating the membranes with specific antibodies using Amersham's ECL chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com).
Quantification In Vivo
Behavioral Tests. Each day, string and pole tests were evaluated for each animal by the number of falls per 5 trials. In motility and posture evaluation, each animal received a score of 0 (no effect), 1 (mild effect), or 2 (severe effect) according to the table adapted from [12] in the 5 behavioral categories mentioned above (see Table in supplementary material online). These 5 scores were added to obtain the additive (total out of 10) behavioral score for each animal and averaged over the pertinent experimental group.
Histological Cell Counts. The expression of NeuN in host cells were quantified on blind-coded slides using an inverted Nikon Eclipse microscope (Nikon USA, http://www.nikon.com), with a Hamamatsu C4742-95 digital camera (Graftek Imaging, Inc., Austin, TX, http://www.graftek.com) controlled by Prairie Technologies software (Middleton, WI, http://www.prairie-technologies.com), with an image relay to a desk top computer and a motorized X-Y stage. The striatal area was first identified under a 2x objective and then sampled under a 40x objective by moving the counting frame systematically through the striatal region using the motorized X-Y stage. The Metamorph offline software (Universal Imaging, West Chester, PA, http://www.microscopy.info) was used to automatically count and average cell numbers. The counts from the area of both striata (as indicated by the slashed area in drawing in Fig. 3) were performed on sections from the 4 coronal levels 180, 200, 210, and 220 as defined in [13]. This covered the area of maximal lesioning, also described in [11]. The combined numbers of labeled cells from the 4 striatal levels obtained from left and right hemisphere were then compared within an animal or between the experimental groups and expressed as mean percentage values ± SEM in comparison to intact controls.
Quantification In Vitro
All quantifications of cell cultures were done in a double blind fashion, using a Zeiss Axioplan 2 microscope (Carl Zeiss) and images were captured through a 40x objective using Zeiss AxioCam digital camera (Carl Zeiss) and Axiovision 4 software. Per evaluated time point, n equaled 12–16, with counts recorded over 15–20 visual fields per experiment and from 4 consecutive experiments, each comprising at least 3 independent, parallel cultures per culture paradigm and assay.
Statistical Analysis
All data are expressed as mean ± SEM, and statistical significance was determined by analyses of variance with Dunnett's post hoc test for multiple comparisons with the control, or Bonferroni's multiple comparison test for multiple comparisons between treatment groups (see also the figure legends describing specific results). Significance of single comparisons was determined in a Student's t-test or the Welch-corrected unpaired t-test, where appropriate. Differences were accepted as significant at p < .05 or less.
Chemicals
3-NP was obtained from Sigma (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com); CM-H2DCFDA, MTT, and Hoechst 33342 were obtained from Molecular Probes (Eugene, OR, http://www.probes.com). All the cell culture supplies were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). Antibodies against the following markers were purchased from: nestin, NeuN, MAP2ab, Calb, BDNF, CNTF, GDNF, and NT-3 from Chemicon (Millipore, Temecula, CA, http://www.millipore.com); GFAP and S100β from Sigma (St Louis); Tuj1 from Covance (Berkeley, CA, http://www.covance.com); VEGF from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com); HNE from Alpha Diagnostic International (San Antonio, TX, http://www.4adi.com) and SOD2 from Upstate (Lake Placid, NY, http://www.upstate.com).
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RESULTS |
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Changes in the following recordings were the most striking: peak values (the higher the value, the worse the symptoms), length of the recovery period, and degree of recovery. Animals without NSP implants [dashed lines] were significantly (p < .001 compared to intact controls) more vulnerable to 3-NP (much higher scores on deficit scale, longer and incomplete recovery), than NSP-grafted mice (p < .01 in comparisons between vehicle-injected and NSP-grafted animals). The latter [solid lines with filled circles], when compared to the mock-grafted groups [dashed lines with filled and empty triangles], demonstrated a decrease in peak behavioral scores of over 50% in all the 3 tests.
Striatal Cytoarchitecture in Mock-Grafted Controls
Three days after 3-NP treatment, animals presented pale (due to reduced hematoxylin stain) lesion areas (dia. between 350 and 450 µm) in the dorso-lateral striata (Fig. 2Aa–2Ad), surrounded by astrocytes and containing many pyknotic and necrotic cells [arrows in Fig. 2Bb]. The number of striatal neurons, assessed by NeuN-staining, was reduced by about 35% in both hemispheres (Fig. 3Aab, black columns, p < .01 compared to intact controls) and a subsequent immunodetection of accumulating HNE, a product of lipid peroxidation in cell membranes, revealed the presence of oxidative stress in these damaged areas (Fig. 3Bb).
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Striatal Cytoarchitecture in NSP Grafted Animals
Three days after intoxication of the host, the proactively grafted NSPs still remained relatively close to the injection site (Fig. 2Ba and represented by the black dots in coronal slice drawings in Fig. 2A). By 3 weeks post-3-NP, they had dispersed within the ipsilateral striatum and were also observed in the corresponding contralateral area (represented by the black dots in coronal slice drawings in Fig. 2A). Only rarely were they found in other brain regions.
At both evaluated time intervals, the striatal tissue ipsilateral to the NSP deposit remained spared from the appearance of cell depleted areas (Fig. 2Aefij) and no elevated HNE-positivity was present (Fig. 3Bc). While the damage of the contralateral (non-grafted) hemisphere appeared similar to that of the mock grafted animals (Fig. 2Agh) it likewise diminished substantially 3 weeks post-3-NP (Fig. 2Akl). Correspondingly, the numbers of striatal neurons ipsilateral to the NSP deposit always remained at almost intact values (Fig. 3Aa, p < .01 compared vehicle injected controls) while cell loss was pronounced and stable in the nongrafted hemisphere (Fig. 3Ab, p < .01 compared to intact controls). Grafting of NSPs into intact control mice had no effects.
The above semiquantitative results need to be confirmed with more rigorous stereological cell counts. Nevertheless, the presented behavioral and first histological data allow the hypothesis that the grafted NSPs exerted a neuroprotective effect on the host tissue, resulting in significant reduction of oxidative damage. In connection with the latter, we did not observe any overt inflammation related or tissue defects, which may have suggested a significant contribution of macrophages and microglia to the observed phenomena (partly confirmed in our sections stained with hematoxylin and ED1 [CD68]-specific antibodies recognizing activated microglia). This, however, has to be followed up with a more detailed study using additional cell and cytokine specific antibodies. Apparently, the presence of NSPs allowed faster behavioral recovery while a natural redundancy of striatal neural circuitry, probably also responsible for the spontaneous but much slower recovery of the mock-grafted mice, may have compensated for the ensuing imbalance of neuronal numbers. This also needs to be addressed in additional experiments able to unmask more subtle interhemispheric differences in motor control and the corresponding behavioral effects (reaction time, precision of movement, etc.).
Coculture with NSPs and the Resistance of PCs to Oxidative Stress
Based on our in vivo experiments, we continued our subsequent investigations in vitro in order to study, in a more controlled and reproducible environment, the molecular bases of the neuroprotective interaction between grafted and host cells after 3-NP exposure. Guided by the histological data obtained, we decided to focus at first on antioxidant mediated events and related growth factor signaling and established an NSP/PC coculture paradigm wherein NSP induced neuroprotection could be studied. At first, the vulnerability of PCs to 3-NP was tested in an initial titration of the toxin in cultures of primary neuronal and glial cells [PNGs, Fig. 4Ab] and cultures of primary glia [astrocytes, PGs, Fig. 4Ad]. Both proved themselves more resistant to the toxin than primary neuronal cultures [PNs, Fig. 4Ac]. Therefore, in order to elicit the same effects in all three culture types, PNGs were treated with 0.1 mM 3-NP, PGs with 0.25 mM, and PNs with 0.05 mM 3-NP. Treatment of NSPs grown alone [Fig. 4Aa], consequently, had to be tested with the same three 3-NP concentrations before their coculture with PCs.
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Culturing PCs and NSPs in direct contact [Figure 4Bi-l] or in separate compartments [Fig. 4Bm–4Bp] had no obvious effects on the experimental outcome, suggesting major involvement of soluble factors in the mediation of the neuroprotective effect.
3-NP-Induced Upregulation of GF Production In Vitro
To investigate the possible involvement of soluble factors in the above neuroprotective processes, we observed that without the toxin, NSPs produced significant amounts of BDNF, CNTF, and VEGF [Fig. 5Aa–5Ac, 5Ba] in all culture conditions, while PCs, alone or in presence of NSPs, produced either only very low amounts of CNTF and VEGF [PNGs; Fig. 5Bcd and 5Cb, hatched columns], or amounts too low to detect in our assays [PNs; Figure 5Bef and Ce, hatched columns]. Three NP treatment upregulated both factors in NSP cultures (Fig. 5Bab, p < .05 compared to untreated controls) while PCs alone showed negligible changes in the levels of these factors [Fig. 5Bc–5Bf]. In 3-NP exposed NSP/PC-cocultures, however, a strong upregulation of all 3 factors could be recorded in the stem cells (Fig. 5Cad, white columns, p < .05 compared to untreated controls) and of CNTF in the primary astrocytes (Fig. 5Ad–5Af and 5Cb, white columns, p < .001 compared to untreated controls) in the NSP/PNG cocultures. No change in these factors was recorded in NSP cocultured neurons [Fig. 5Ce, white columns].
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Inhibition of GFs in NSP/PC Cocultures and Its Effects on the Response of the Cells to Oxidative Stress
The functional relationship between the upregulation of GFs and the enhanced resistance of the NSP/PC cocultures to 3-NP, was evaluated in antibody blocking assays. Having correlated changes in ATP levels with measurements of MTT, free radical, and apoptosis [Fig. 4], in the following, we concentrated on the last 3 only to describe the behavior of our cultures.
While an interference with BDNF activity had no significant effect [Fig. 6B], both the mitochondrial activity and levels of ROS in PNGs and PNs progressively deteriorated after inhibition of CNTF (Fig. 6C, p < .01 compared to uninhibited controls) or VEGF (Fig. 6D, p < .01 compared to uninhibited controls). This led to gradually increasing numbers of apoptotic cells in the cultures, until both PNGs and PNs were unable to recover from the neurotoxic effects as compared to noninhibited controls (Fig. 6, compare lowermost panels in C and D with that in A). A simultaneous neutralization of all 3 tested GFs led, not surprisingly, to the most severe deterioration of coculture viability and approached it to that of the 3-NP-treated PCs cultured alone (compare E and F in Fig. 6, p < .001 compared to uninhibited controls in column E). In most measurements, interestingly, PNG viability [bold lines] appeared to deteriorate less severely (p < .05) than that of PNs [thin lines].
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GF Production and Upregulation of Antioxidant Enzymes In Vitro
The reduced cell viability and increased ROS production in 3-NP-treated cocultures after growth factor inhibition suggested that these factors may have directly participated in the upregulation of cellular antioxidative defense mechanisms. To explore this possibility, we monitored by immunocytochemistry and in Western Blots NSC-mediated changes of intracellular SOD2 levels. We focused on SOD2 since it is known to be inducible by VEGF [16, 17], is an important member of the antioxidant defense system in mammalian cells and has been shown to play an important role in quenching of oxidative stress in the striatum [18–20].
NSPs themselves reacted to 3-NP exposure by increased SOD2 expression [Fig. 7A] which was in harmony with their initially found resistance to the toxin [8]. Interestingly, a similar upregulation of this enzyme was also induced in PNGs and PNs in coculture with NSPs (Fig. 7, microphotographs in Ba and Ca and lanes 3 in the blots in Bb and Cb). Although Figure 7 shows the upregulation of SOD2 at 48 hours after the 3-NP treatment, at which point a strong protective effect was observed [Fig. 4], it's increased presence was already noted immediately after removal of 3-NP, i.e., the zero time point in the graphs in Figure 1 (data not shown). On the other hand, no changes in SOD2 were found in untreated cocultures and 3-NP-treated PCs alone [Fig. 7, lanes 1 and 2 in blots in Bb and Cb].
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Upregulation of SOD2 and Prevention of Oxidative Stress In Vivo
Having found in vitro that GF mediated upregulation of SOD2 was involved in the observed neuroprotective influence of NSPs on their cellular environment, we decided to go back to our in vivo model and to ask whether changes in expression of this antioxidant could also be detected in our graft protected 3-NP-treated mice. Interestingly, in mock-grafted animals, endogenous SOD2 production was insignificant at 3 days after 3-NP treatment and, accordingly, a strong HNE specific staining revealed oxidative damage [Figure 7Db, see Figure 3Bb for HNE]. In NSP-grafted, 3-NP-treated animals, on the other hand, we observed a strong expression of SOD2 in grafted and host cells, the latter including neurons [Fig. 7Dcd]. Although these data need to be confirmed with a confocal analysis, they were nevertheless in harmony with our findings in vitro and positively correlated the upregulation of GF expression with increased activity of an important antioxidant defense mechanism. The resulting reduction in oxidative stress was reflected in down-regulation of the HNE signal, which now became comparable to that of intact animals (compare Fig. 3Ba and 3Bc). The elevated SOD2 activity disappeared by 3 weeks post-3-NP, regaining base line levels found in intact animals.
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DISCUSSION |
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Recently, it has been demonstrated that exogenous stem cells can create host micro-environments favoring recovery or preservation of damaged or imperiled cells [1, 6, 21–24]. This return to homeostasis is many times accompanied by the intrinsic production and secretion of donor NSP derived GFs, including GDNF, NT-3, BDNF, and NGF [1, 6, 22, 24, 25]. As demonstrated in this study and our previous work [8], the baseline expression of certain GFs in NSPs can be substantially higher than that of mature neural cell types and can increase more vigorously upon arrival of oxidative stress. This "stemness feature" would appear crucial for the usefulness of NSPs in neurotransplantation since they need to resist the hostile environment of the diseased nervous system in order to exert their beneficial influence [8].
The presented experiments not only demonstrated the donor NSPs to be a major source of GFs like BDNF, CNTF, and VEGF, but they revealed for the 1st time a collaborative interaction between NSPs and the surrounding differentiated cell types. The NSP dependent recruitment of astrocytes and their increased production of CNTF resulted in higher resistance of PNGs and PGs to 3-NP when compared to PNs. The fact that NSP/PG cocultures appeared to be slightly less protected than cultures of NSPs with PNGs (even though the differences proved statistically insignificant) may have reflected the greater struggle of the NSPs themselves in presence of the elevated 3-NP concentration required in the cocultures with PGs. As described recently, astrocytes possess a variety of homeostasis regulating properties and represent important and influential components in the stem cell niche [26–28]. Here, we may assign to them a new capability, namely, responsiveness to NSP derived GFs in presence of injury, resulting in a paracrine enhancement of their own secretion of neuroprotective factors.
We know that NSPs, neurons, and glial cells do all express receptors for CNTF and VEGF [29–33] which makes the above mentioned interactions plausible. Another important quality of VEGF and CNTF is their involvement in controlling cellular resistance against oxidative stress. While CNTF promotes cellular resistance to stress stimuli by activation of the JAK/STAT and MAPK pathways [34, 35], VEGF can be regulated by cellular redox processes and oxidative stress [36] and is apparently influencing the production of cellular SOD2 [16, 17, 37] Thus, through participation in the upregulation of SOD2, VEGF was likely to contribute to the observed neuroprotection as well and we decided to concentrate our efforts on this factor (pathways involved in CNTF-mediated neuroprotection will be addressed in future studies).
Both in vitro and in vivo, we found an involvement of SOD2 in the cellular response to 3-NP exposure. Not only did the NSPs strongly express SOD2, but their presence was associated with an induction of the antioxidant in the surrounding cells, including neurons. In vivo, this resulted in efficient maintenance of local tissue homeostasis, apparent by the reduced striatal HNE staining, and thus corroborated other studies suggesting SOD to play an important antioxidant role in the striatum [18–20]. The correlation of enhanced cellular production/secretion of CNTF and VEGF with upregulation of SOD2 in the NSP mediated cellular networks in vitro together with the increased SOD2 expression in the NSP grafted 3-NP-treated mice suggested the direct role of these molecules in the elevated cellular resistance of NSPs and the other neural cells to 3-NP-induced oxidative stress. We tested this hypothesis by blocking the action of VEGF with neutralizing antibodies in the cell cultures. Intriguingly, the resulting elimination of cell survival capability (a similar effect was also observed after blocking the action of CNTF) was indeed accompanied by a marked loss of SOD2 expression and pointed towards a direct participation of VEGF in signaling events triggering an upregulation of this superoxide detoxifier.
Obviously, it has still to be demonstrated that both CNTF and VEGF also directly regulate SOD2 expression in vivo. Although a promising result, this causal relationship will have to be looked at in more detail by, for example, blocking the action of the GFs in specific RNAi experiments while monitoring changes in SOD2 expression. Genetically modified primary cells or animals compromised in the production of these factors or in SOD2 production may be another option.
In conclusion, this study provides evidence that direct activation and control of antioxidant defense mechanisms through GF signaling appears to accompany NSP mediated neuroprotection. NSPs, can thereby act as cellular "probes" and initiate an appropriate network of GF producing cells, including themselves and resident elements like astrocytes. Whether GFs alone or cell types other than NSPs producing them would result in a similar effect, we don't know. However, NSPs, by their neural origin, multipotency, relative resistance to oxidative stress, and their inherent capacity to adapt the production of GFs according to pathology [6, 25] might still be better candidates for intracellular communication in the CNS. A deeper understanding of the novel "recruiting" characteristic of NSPs and the role of the various GFs and the involved signaling pathways might thus add another important element to the kaleidoscope of NSP features useful for treatment of neuropathologies.
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ACKNOWLEDGMENTS |
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  D. Yu, W. L. Neeley, C. D. Pritchard, J. R. Slotkin, E. J. Woodard, R. Langer, and Y. D. Teng Blockade of Peroxynitrite-Induced Neural Stem Cell Death in the Acutely Injured Spinal Cord by Drug-Releasing Polymer Stem Cells, May 1, 2009; 27(5): 1212 - 1222. [Abstract] [Full Text] [PDF]
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 N. D. Bull, G. A. Limb, and K. R. Martin Human Muller Stem Cell (MIO-M1) Transplantation in a Rat Model of Glaucoma: Survival, Differentiation, and Integration Invest. Ophthalmol. Vis. Sci., August 1, 2008; 49(8): 3449 - 3456. [Abstract] [Full Text] [PDF]
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: Expression detectable but too low to be graphically visualized. Bars: 20 µm (Aa–Ac), 40 µm (Ad–Af).
-tubulin. Data were obtained with proteins isolated in 3 separate experiments. NSP dependent upregulation of SOD2 could also be observed with preliminary immunostaining in vivo. In comparison to intact (Da) and vehicle-grafted-3-NP-treated controls (Db), 3 days after 3-NP treatment, an NSP-mediated up-regulation of the anti-oxidant enzyme SOD2 was observed in both host [red in Dc] and double stained donor cells [yellow in Dc]. About 60% of NeuN-positive host cells within the grafted area also expressed SOD2 [red in Dd]. These SOD2 changes within the animal groups correlated well with the variations in HNE staining illustrated in