Brain Advance Access published online on May 30, 2008
Brain, doi:10.1093/brain/awn096
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis
1Institute of Neuropathology, University Hospital Münster, Münster, Germany, 2Department of Neuropathology, University Medical Centre Göttingen, 3Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Canada and 4Institute for Multiple Sclerosis Research, University Medical Centre Göttingen and Gemeinnützige Hertie-Stiftung, Göttingen, Germany
Correspondence to:
Tanja Kuhlmann, Institute of Neuropathology, University Hospital Münster, Münster, Domagkstr. 19, 48149 Münster, Germany E-mail: tanjakuhlmann{at}gmx.de
 |
Summary |
|---|
 Top Summary IntroductionMaterial and MethodsResultsDiscussionReferences |
|---|
Impaired function/differentiation of progenitor cells might provide an explanation for the limited remyelination observed in the majority of chronic multiple sclerosis lesions. Here, we establish that in the normal adult human CNS, the transcription factors Nkx2.2 and Olig2 are strongly expressed in progenitor cells while mature oligodendrocytes are characterized by low levels of Olig2 or Nkx2.2. In vitro studies confirmed the expression of Olig2 in oligodendroglial progenitor cells and mature oligodendrocytes while astrocytes, microglial cells and neurons were negative for Olig2. In early multiple sclerosis lesions, we found Olig2-positive progenitor cells throughout all lesion stages and in periplaque white matter (PPWM). The number of progenitors in PPWM was significantly increased compared with the white matter from controls. In chronic multiple sclerosis lesions progenitor cells were still present, however, in significantly lower numbers than in early multiple sclerosis lesions. A subpopulation of progenitor cells in early multiple sclerosis lesions and PPWM but not in control cases co-expressed NogoA, a marker of mature oligodendrocytes. The co-expression of these two markers suggested that these cells were maturing oligodendrocytes recently recruited from the progenitor pool. In contrast, in chronic multiple sclerosis lesions maturing progenitors were only rarely present. In summary, we provide evidence that a differentiation block of oligodendroglial progenitors is a major determinant of remyelination failure in chronic multiple sclerosis lesions.
Key Words: oligodendroglial progenitors; multiple sclerosis; Olig2; Nkx2.2
Abbreviations: OPCs, oligodendroglial progenitor cells; PPWM, periplaque white matter
Received December 5, 2007. Revised April 15, 2008. Accepted April 25, 2008.
|
Introduction |
|---|
|
TopSummaryIntroductionMaterial and MethodsResultsDiscussionReferences |
|---|
Multiple sclerosis is the most frequent demyelinating disease of young adults. Multiple sclerosis lesions are characterized by demyelination, inflammation, axonal loss and reactive gliosis. The myelin maintaining mature oligodendrocytes are a presumed target of the immune mediated demyelinating process in this disease. Oligodendrocyte loss is pronounced in a subset of patients early in the disease process (Brück et al., 1994
; Ozawa et al., 1994; Lucchinetti et al., 1999) and may even represent the initial event in lesion formation (Barnett and Prineas, 2004). However, concurrently with these destructive processes, repair mechanisms are initiated in these lesions. Remyelination can be observed in lesions with ongoing demyelinating activity (Prineas et al., 1993; Raine and Wu, 1993). Analyses of chronic multiple sclerosis lesions indicate that although remyelination may be extensive in some of them, it is absent or limited to the plaque border in the majority (Prineas and Connell, 1979; Barkhof et al., 2003; Patrikios et al., 2006; Patani et al., 2007). The underlying cause for this limited remyelination is not known. Possible explanations include a lack of oligodendroglial progenitor cells (OPCs), an impaired differentiation of this cell population and/or disturbed interaction between myelinating cells and axons.
Remyelination of CNS lesions has been shown in animal models to be dependent on the recruitment and differentiation of OPCs (Targett et al., 1996; Keirstead and Blakemore, 1997). Increased numbers of OPCs and mature oligodendrocytes have been found in areas of ongoing remyelination in early multiple sclerosis lesions suggesting OPC recruitment and/or differentiation (Raine et al., 1981; Lucchinetti et al., 1999). However, recent publications indicate that the number of OPCs is reduced over time in multiple sclerosis lesions (Chang et al., 2002; Reynolds et al., 2002; Wolswijk 2002; Wilson et al., 2006). In mice repeated episodes of ethidium bromide-induced demyelination with intervening recovery did not affect the extent of remyelination nor the numbers of OPCs (Penderis et al., 2003). In contrast, mice which underwent chronic demyelination over an extended period of time showed progressive depletion of OPCs and lack of remyelination (Mason et al., 2004).
Studies addressing the fate of OPCs in human demyelinating diseases in early versus late disease stages have been limited by the lack of antibodies recognizing these cells in human paraffin embedded tissues as well as the relatively small number of available human tissue specimens from early disease stages. Antibodies frequently used to identify OPCs, such as NG2 and PDGF-receptor (R)
, have until now only successfully been applied to frozen and not human paraffin embedded human tissue sections. Furthermore, NG2 may also label other glial cell populations, such as synantocytes (Butt et al., 2002; Wilson et al., 2006). Additional markers or a combination of markers identifying OPCs in human paraffin embedded tissue sections are therefore desirable.
Olig2 and Nkx2.2 are transcription factors expressed by OPCs and mature oligodendrocytes. Recent studies have revealed the importance of Olig2 for the differentiation of neural progenitor cells into the oligodendroglial lineage (Lignon et al., 2006) while Nkx2.2 regulates the maturation and differentiation of oligodendroglial progenitors (Qi et al., 2001). Overexpression of Olig2 induces differentiation of neural stem cells into mature oligodendrocytes in vitro (Copray et al., 2006). Disruption of Olig2 leads to an ablated formation of oligodendrocytes in the spinal cord and lack of NG2 cells (Lu et al., 2002; Lignon et al., 2006). In contrast, loss of Nkx2.2 results in increased numbers of PDGF-R-positive oligodendrocytes (Qi et al., 2001). Strong Olig2 or Nkx2.2 signals were observed in OPCs in the spinal cord of adult mice, while mature oligodendrocytes expressed low levels of these two transcription factors (Kitada and Rowitch, 2006).
In this report, we demonstrate that the transcription factors Olig2 and Nkx2.2 identify progenitors in human adult CNS. In early multiple sclerosis lesions, increased numbers of progenitor cells were observed in periplaque white matter (PPWM) compared with white matter from controls. In chronic multiple sclerosis lesions, OPCs were still present, albeit in lower numbers. While about one-third of the progenitor cells in early multiple sclerosis tissue specimens were in the transition to mature oligodendrocytes, only rare individual maturing progenitors were observed in chronic multiple sclerosis lesions—suggesting an impaired ability of OPCs to differentiate over time in multiple sclerosis lesions. Our data provide evidence that a differentiation block of OPCs contributes to limited remyelination observed in chronic multiple sclerosis lesions.
|
Material and Methods |
|---|
|
TopSummaryIntroductionMaterial and MethodsResultsDiscussionReferences |
|---|
Tissue samples
We retrospectively investigated brain tissue from 43 multiple sclerosis patients and five control cases. Biopsies (n = 33) had been performed for diagnostic reasons to exclude neoplastic or infectious diseases. Informed consent had been obtained from each patient. None of the study authors was involved in decision-making with respect to biopsy. For 18 patients detailed information was available (sex, age, duration between first symptom of index lesions to biopsy) (Table 1). In addition to the biopsy cases, 11 tissue blocks with 12 lesions from 10 autopsy cases were analysed. The disease duration varied between 4 and 34 years (Table 1). Control tissue specimens were derived from six patients who underwent temporal or occipital lobe surgery due to pharmaco-resistant seizures. The tissue specimens of these control patients revealed unspecific changes such as focal microglial activation and reactive astrogliosis. The study was approved by the Ethics Committee of the University of Göttingen.
|
In vitro studies
Human foetal oligodendrocyte progenitor culture
Human foetal CNS tissue of 16- to 21-week-old embryos was provided by the Human Fetal Tissue Repository (Albert Einstein College of Medicine, Bronx, NY). The studies were approved by the institutional review boards of McGill University and the Albert Einstein Institute. Human foetal OPCs were isolated as previously described (Miron et al., 2008
). Briefly, brain tissue was mechanically diced, incubated with 0.25% trypsin (Invitrogen) and 25 µg/ml DNase I (Roche, Laval, QC) at 37°C and washed through a 132 µm nylon mesh (Industrial Fabrics, Minneapolis, MN). OPCs were isolated by immunomagnetic bead selection according to the manufacturer's instructions using A2B5 IgM antibody purified from hybridoma (neat) and rat anti-mouse IgM antibody (Miltenyi Biotech, Auburn, CA). The A2B5+ cell fraction was resuspended in DMEM-F12 supplemented with 1% penicillin–streptomycin, 1% glutamine (all from Invitrogen, Karlsruhe, Germany), N1 supplement (1X; Sigma), thyroid hormone (T3, 2 ng/ml; Sigma-Aldrich, Munich, Germany), basic fibroblast growth factor (bFGF, 20 ng/ml; Sigma-Aldrich, Munich, Germany) and PDGF (20 ng/ml; Sigma-Aldrich) and plated on lysed cultures of human foetal astrocytes for survival purposes. Cultures were maintained at 37°C for 10 days before fixation. Our previous studies have characterized the purity of these cultures (Miron et al., 2007, 2008).
Human adult mature oligodendrocyte culture
Temporal lobe surgical resections from patients treated for non-tumour related intractable epilepsy were obtained in agreement with the guidelines of the Biomedical Ethics Unit of McGill University. Mature oligodendrocytes were isolated as previously described (Yong and Antel, 1992). Tissue was digested with 0.25% trypsin and 25 µg/ml DNase I, mechanically dissociated through a 132 µm nylon mesh and separated on a linear 30% Percoll density gradient (GE Healthcare, Uppsala, Sweden). Myelin and red blood cells were removed and neural cells were plated in uncoated tissue culture flasks overnight in minimal essential culture medium containing 5% foetal calf serum, 1% penicillin–streptomycin, 1% glutamine, 0.1% glucose (all from Invitrogen, Karlsruhe, Germany). Through two rounds of differential adhesion, less adherent cells (including oligodendrocytes) were harvested and plated in poly-L-lysine coated 16 well glass chamber slides at a density of 105 cells per well. Adherent cells (microglia) were plated in uncoated plastic eight well chamber slides at a density of 5 x 104 cells/well. The purity of these cultures has been previously characterized (D'Souza et al., 1996; Wosik et al., 2004).
For isolation of oligodendrocyte progenitor cells from these cultures, the less adherent cells were submitted to immunomagnetic bead separation as described earlier.
Flow cytometry
Ex-vivo human A2B5 + and A2B5– foetal brain cells were washed in staining buffer (PBS with 1% FCS and 0.1% sodium azide), incubated with A2B5 antibody (1 : 100) or isotype IgM control (1 : 10, Dako, Glostrup, Denmark), then washed twice with buffer and incubated with goat anti-mouse secondary antibody conjugated to phycoerythrin (BD Biosciences, Mississauga, ON, Canada). Following fixation in 1% formaldehyde, results were acquired using a FACSCalibur (BD Biosciences) and analysed using FlowJo software (Treestar, Ashland, USA). The positive fraction was typically >90% A2B5 +.
Immunocytochemistry
Cells were fixed with 2% paraformaldehyde and permeabilized with cold methanol for myelin marker stains (MAG, MBP), acetic alcohol for the GFAP stain and 0.25% triton X-100 for the βTubIII, NogoA and Olig2 stains. Cells were blocked with HHG (1nM HEPES, 2% horse serum, 10% goat serum, in Hank's balanced salt solution) and primary antibodies were applied for 1 h at 4°C. Mouse monoclonal primary Abs used include A2B5 (1 : 100), O4 (1: 100; Chemicon International, Temecula, CA), GFAP-Alexa-488 (1 : 100; Molecular Probes, Burlington, Canada), βTubIII (1 : 400; Sigma), myelin-associated glycoprotein MAG (1 : 200; Chemicon International, Temecula, CA), NogoA (1 : 1000; generous gift from Dr Martin Schwab, University of Zurich) and myelin basic protein MBP (1 : 500; Sternberger Monoclonals Inc., Lutherville, MD). Rabbit primary antibodies include NogoA (1 : 100; Chemicon International, Temecula, CA) and Olig2 (1 : 50; IBL, Spring Lake Park, Minnesota). Cultures were incubated with the appropriate secondary antibodies for 30 min at 4°C : goat anti-mouse secondary antibody conjugated to Cy3 (1 : 500; Jackson ImmunoResearch, Westgrove, PA) and goat anti-rabbit conjugated to fluorescein isothiocyanate fluorochrome (FITC) (1 : 100; Cedarlane, Burlington, ON, Canada). Hoechst dye was used to label nuclei (bis-benzimide, 1 : 1000; Molecular Probes). Control stainings with isotypes corresponding to primary antibodies showed low non-specific staining. Slides were mounted using Fluoromount-G (Southern Biotech, Birmingham, AL). Epifluorescent microscopy (Leica, Wetzlar, Germany) was used to visualize stains using OpenLab imaging software.
Classification of multiple sclerosis lesions
All lesions fulfilled the generally accepted criteria for the diagnosis of multiple sclerosis (Prineas, 1985; Allen, 1991; Lassmann et al., 1998). Demyelinating activity was classified as described in detail earlier (Brück et al., 1995). In this study, we focused on actively demyelinating, demyelinated and remyelinating lesion areas. Actively demyelinating lesion areas (biopsies: n = 17, autopsies: n = 0) were located at the plaque border, these areas were partially demyelinated and infiltrated by numerous macrophages containing myelin degradation products within their cytoplasm. Demyelinated plaques were infiltrated by macrophages and T cells, but macrophages did not contain myelin degradation products (biopsies: n = 5, autopsies: n = 10). In remyelinating plaque areas (biopsies: n = 11, autopsies: n = 3), thin, irregularly formed myelin sheaths were seen. Remyelinating lesions early during disease course were infiltrated by numerous macrophages and T cells while remyelinating plaques from chronic multiple sclerosis are characterized by a very mild inflammatory reaction. PPWM (biopsies: n = 15, autopsies: n = 12) showed no signs of demyelination.
Immunohistochemistry
Biopsies were performed in different centres all over Germany and sent to the Department of Neuropathology in Göttingen after completion of routine analyses. Specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Autopsy cases were derived from a collection of multiple sclerosis autopsies collected at the Montreal Neurological Institute, McGill University, Montreal, Canada. Biopsy and autopsy tissues were cut in 4 µm thick sections that were stained with haematoxylin and eosin, Luxol-fast blue and Bielschowsky's silver impregnation. Immunohistochemical staining was performed with an avidin-biotin technique. After deparaffinization intrinsic peroxidase activity was blocked by incubation with 5% H2O2 in PBS for 20 min. Non-specific antibody binding was inhibited with 10% FCS in PBS for 25 min. For biopsies microwave pre-treatment for better antigen retrieval was performed for mouse anti-Nogo-A, rabbit anti-Nogo-A, rabbit anti-Olig2, rat-anti-CD3 and mouse anti-Nkx2.2. Autopsy cases were pre-treated with Triton X (1% for 1 h) prior to incubation with anti-Olig2 and anti-NogoA antibodies. The primary antibodies were rabbit anti-myelin basic protein (1 : 1000) (Boehringer Mannheim, Mannheim, Germany), mouse anti-KiM1P (1 : 5000) (H.-J. Radzun, Department of Pathology, University of Göttingen, Germany), rat anti-CD3 (1 : 200) (Serotec, Oxford, UK), rabbit anti-GFAP (1 : 2000) (Dako, Denmark), mouse anti-GFAP (1 : 50) (Dako, Denmark), mouse anti-NeuN (1 : 100) (Chemicon International, Temecula, CA), rabbit anti-Olig2 (1 : 300) (IBL, Spring Lake Park, Minnesota), mouse anti-Nkx2.2 (1 : 100) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa), rabbit anti-Nogo-A (1 : 750) (Chemicon International, Temecula, CA) and mouse anti-Nogo-A (1 : 15 000) (11c7, a generous gift from M.E. Schwab, Brain Research Institute, University of Zürich and Department of Biology, Swiss Federal Institute of Technology Zürich, Switzerland). Secondary antibodies were anti-mouse, anti-rabbit or anti-rat biotinylated Ig (1 : 200) (Amersham Biosciences, Freiburg, Germany). For double stainings, we combined the rabbit anti-Olig2 or mouse anti-Nkx2.2 antibodies with the mouse or rabbit anti-NogoA antibody, mouse or rabbit anti-GFAP, mouse anti-NeuN or mouse anti-KiM1P. Secondary antibodies were conjugated to either Cy2 or Cy3 (1 : 200) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA).
Morphometry and statistics
The number of Nogo-A, Olig2 and Nkx2.2-positive cells stained with the corresponding antibodies was determined in at least 10 standardized microscopic fields of 10 000 µm each defined by an ocular morphometric grid. In the text and figures, the mean number of cells/mm2 ± SEM is given. The numbers of weakly and strongly positive Olig2- and Nkx2.2-positive cells in controls were determined by two independent observers. The intensity of the Olig2 and Nkx2.2 signals in selected control and multiple sclerosis cases was measured using the Image J program confirming that the strongly and weakly Olig2 and Nkx2.2 cells were indeed two distinct cell populations. For statistical analysis, Student t-tests were performed. All tests were classified as significant if the P-value was < 0.05. The GraphPad PRISMTM software was used (Graph Pad Software, Inc., San Diego, CA, USA) for these analyses.
|
Results |
|---|
|
TopSummaryIntroductionMaterial and MethodsResultsDiscussionReferences |
|---|
Expression of Olig2 and Nkx2.2 in adult human CNS tissue
In adult mouse CNS tissues Olig2 and Nkx2.2 are expressed weakly in mature oligodendrocytes and strongly in progenitor cells (Kitada and Rowitch, 2006
). To address the question whether Olig2 and Nkx2.2 show a similar expression pattern in adult human CNS tissues, we stained six control biopsies from patients who underwent brain surgery due to pharmaco-resistant seizures with anti-Olig2 and anti-Nkx2.2 antibodies. Olig2 and Nkx2.2-positive cells were found in white matter and cortex (Fig. 1A–D); as expected, significantly higher numbers were found in the white matter (Fig. 1A–D). The majority of cells in the white matter showed a weak nuclear Olig2 (Olig2weak 995 ± 173 cells/mm2) or Nkx2.2 signal (Nkx2.2weak 968 ± 78 cells/mm2) (second observer: Olig2weak 924 ± 94 cells/mm2, Nkx2.2weak 1070 ± 113 cells/mm2). As expected, the total numbers of weakly labelled Olig2- and Nkx2.2-positive cells in the cortex were lower than in white matter (Olig2weak: 181 ± 24 cells/mm2; Nkx2.2 weak cells: 228 ± 31 cells/mm2) (Fig. 1E and F). The distribution pattern of strongly stained Olig2- and Nkx2.2-positive cells showed a similar distribution pattern as described for NG2-positive progenitor cells (Butt et al., 2002). In grey and white matter, we found comparable numbers of strongly Olig2- or Nkx2.2-positive cells, respectively (Olig2strong white matter: 46 ± 2 cells/mm2, Olig2strong cortex: 74 ± 9 cells/mm2; Nkx2.2strong white matter: 34 ± 7 cells/mm2, Nkx2.2strong cortex: 37 ± 3 cells/mm2) (second observer Olig2strong white matter: 36 ± 2, Nkx2.2strong white matter: 30 ± 1) (Fig. 1E and F). To clarify whether the same cell populations strongly express Olig2 and Nkx2.2, the appropriate double immunohistochemical staining was conducted. Not all Olig2strong cells also expressed Nkx2.2 strongly (Fig. 2A–C) as expected from our quantitative analysis of the numbers of Nkx2.2strong and Olig2strong cells in white matter and cortex. These findings indicate either the presence of different differentiation stages of progenitors in the human CNS or a heterogeneity among progenitor cells, as suggested for the rodent CNS (Kitada and Rowitch, 2006).
|
|
Double immunohistochemistry with anti-Nogo-A, a marker that reliably identifies mature oligodendrocytes in human CNS tissue (Kuhlmann et al., 2007
), revealed that almost all of the weakly positive Nkx2.2 or Olig2 cells were also Nogo-A positive and were identified as mature oligodendrocytes, therefore (Fig. 2E and F). We also observed a few reactive astrocytes that expressed low levels of Nkx2.2 in the nucleus (data not shown). In contrast, Olig2strong cells were negative for Nogo-A (Fig. 2F) as well as for the astrocytic, neuronal and microglial markers GFAP, NeuN and KiM1P demonstrating that strong nuclear expression of Olig2 identifies selectively progenitor cells in adult human CNS.
Expression of Olig2 in oligodendroglial linage cells in vitro
To further confirm that expression of Olig2 is restricted to the oligodendroglial lineage, we studied the expression of Olig2 in cells isolated from human foetal and adult CNS in vitro. Foetal CNS derived cells were bead-selected for A2B5 and cultured for 10 days in vitro. Flow cytometry (FACS) analysis confirmed that the bead-selected cultures were highly enriched for A2B5-positive cells (data not shown). In these cultures, we found numerous Olig2-positive cells. Co-labelling with two markers of the oligodendroglial lineage (A2B5 and O4) revealed that all A2B5- and O4-positive cells expressed Olig2 (Fig. 3A and B). However, there were also Olig2-positive cells that were negative for O4 (Fig. 3B). NogoA-positive cells were not detected in foetal cell cultures enriched for A2B5-positive cells confirming earlier studies (data not shown) (Ruffini et al., 2004). However, we also found single GFAP-positive astrocytes and β-tubulin-III-positive neurons in foetal cell cultures, which did not express Olig2 (Fig. 3C and D). Mature oligodendrocytes that were derived from tissue specimens from adult patients undergoing surgery due to intractable epilepsy expressed GalC, MAG, NogoA as well as Olig2 (Fig. 4A–C). In contrast, microglial cells derived from human adult CNS did not show any Olig2 expression (data not shown).
|
|
Progenitor cells in multiple sclerosis
In early multiple sclerosis lesions
Due to the fact that Olig2 recognizes a broader spectrum of progenitor cells than Nkx2.2 in identifying OPCs in adult human CNS, we focussed in the following immunohistochemical studies on Olig2. As in CNS tissue specimens from control cases, we found strongly and weakly labelled Olig2 positive cells in multiple sclerosis lesions (Fig. 5A and B).
|
We quantified the numbers of Olig2strong cells in active demyelinating and remyelinating lesions as well as in PPWM and compared the results with our findings in white matter from control cases (Fig. 3C). In PPWM from multiple sclerosis patients, a significant increase in the number of Olig2strong cells was observed compared with white matter from control cases (WM 40 ± 1 cells/mm2, PPWM 133 ± 24 cells/mm2, P = 0.024). In early multiple sclerosis lesions, significantly higher numbers of Olig2strong cells were found in PPWM (133 ± 24 cells/mm2) compared with active demyelinating (40 ± 10 cells/mm2, P = 0.0007) and demyelinated lesions (44 ± 11 cells/mm2, P = 0.048). To analyse whether the Olig2-positive cells proliferate, we conducted double immunohistochemical stainings for Olig2 and the proliferation marker Ki67 in two multiple sclerosis cases characterized by high numbers of Olig2strong cells. Only rare cells were found to co-express Olig2 and Ki67 (data not shown).
In chronic multiple sclerosis lesions
To address whether progenitor cells are still present in chronic multiple sclerosis lesions, we examined the number of Olig2strong cells in multiple sclerosis lesions derived from 10 patients with a long disease duration (between 4 and 34 years). In all patients Olig2strong cells were found in PPWM (67 ± 13 cells/mm2) (Fig. 6A). In demyelinated lesion areas, we found significantly lower numbers of strongly Olig2-positive progenitor cells (26 ± 5 cells/mm2) (Fig. 6B and C). In some demyelinated lesion areas relatively high numbers of Olig2-positive cells were observed despite a complete absence of Nogo-A-positive mature oligodendrocytes. Comparison of the number of Olig2strong cells in early and chronic multiple sclerosis revealed significantly higher numbers of Olig2strong cells in PPWM of early lesions (PPWM early multiple sclerosis 133 ± 24 cells/mm2, PPWM chronic multiple sclerosis 68 ± 13 cells/mm2, P = 0.039), as well as a trend to a higher number in demyelinated lesions areas (DM early multiple sclerosis 44 ± 11 cells/mm2, DM chronic multiple sclerosis 26 ± 5 cells/mm2, P = 0.058) in the early disease stage.
|
Differentiating oligodendrocyte progenitor cells in multiple sclerosis lesions
In tissue specimens from multiple sclerosis patients, we found a third cell population in addition to mature oligodendrocytes (expressing Nogo-A and low levels of Olig2) and progenitor cells (expressing high levels of Olig2 but no Nogo-A) not observed in control cases: strongly Olig2-positive cells that were also Nogo-A-positive (Olig2strong/Nogo-A) (Fig. 7A).
|
In early multiple sclerosis tissue specimens, about one-third of Olig2strong cells also expressed Nogo-A. The highest numbers of Olig2strong/NogoA-positive cells were found in PPWM and remyelinating lesions (PPWM 43 ± 11 cells/mm2, active lesions 19 ± 7 cells/mm2, demyelinated lesions 17 ± 5, remyelinating lesions 37 ± 10 cells/mm2) (Fig. 7B).
In chronic multiple sclerosis Olig2strong/NogoA-positive maturing progenitor cells were found only occasionally in PPWM or in multiple sclerosis lesions (PPWM: 4 ± 2 cells/mm2, DM: 1 ± 1 cells/mm2) as revealed by double-immunohistochemistry (Fig. 7C). To find whether a lack of axons might be responsible for the low numbers of Olig2strong/NogoA-positive cells, we conducted double immunohistochemical stainings for neurofilament and Olig2. Numerous Olig2strong cells were found in close proximity to axons (Fig. 7D).
|
Discussion |
|---|
|
TopSummaryIntroductionMaterial and MethodsResultsDiscussionReferences |
|---|
We demonstrate that in adult human CNS, progenitors can be identified by using a combination of antibodies recognizing oligodendroglial transcription factors (Olig2 and Nkx2.2) together with markers of mature oligodendrocytes (NogoA). In vitro, Olig2 was detected in OPCs and mature oligodendrocytes, while astrocytes, microglial cells and neurons were negative for Olig2. In early multiple sclerosis lesions, the number of progenitors in PPWM was higher than in white matter from controls. In addition, OPCs undergoing transition to mature oligodendrocytes were observed in early lesions but not in controls or in the majority of chronic multiple sclerosis patients. Our data suggest that a reduction of progenitor cells in combination with a differentiation block contributes to the lack of remyelination in chronic multiple sclerosis lesions.
In control CNS tissue specimens, about 5% of Olig2- and Nkx2.2-positive cells showed strong nuclear immunoreactivity and no co-localization with oligodendroglial, astrocytic, microglial or neuronal markers while the remaining Olig2- and Nkx2.2-positive cells displayed a weak nuclear staining. In vitro, we detected Olig2 in OPCs and in mature oligodendrocytes while astrocytes, microglial cells and neurons did not express Olig2. These results demonstrate that Olig2strong and Nkx2.2strong cells in the adult human CNS are progenitor cells as shown for rodent adult CNS (Kitada and Rowitch, 2006). The fact that only a subpopulation of Olig2strong cells also expressed Nkx2.2 at high levels indicates either heterogeneity or the presence of different differentiation stages for progenitor cells in the human CNS as also observed for the rodent CNS (Kitada and Rowitch, 2006). In our study, weakly labelled Olig2- or Nkx2.2-positive cells co-expressed NogoA, a marker for mature oligodendrocytes (Kuhlmann et al., 2007); therefore, we concluded that these cells were mature oligodendrocytes.
In PPWM and in remyelinating early multiple sclerosis lesions, we observed a significantly increased number of strongly Olig2-expressing progenitor cells compared with white matter of control patients. This is in line with earlier reports describing the presence of NG2-, O4- or PDGF-R alpha-positive progenitor cells in multiple sclerosis lesions (Chang et al., 2002; Reynolds et al., 2002; Wolswijk, 2002; Wilson et al., 2006). As observed in early multiple sclerosis lesions, increased numbers of progenitor cells can be found in remyelinating animal models. In mice in which demyelination has been induced by toxins, such as ethidium bromide, increased numbers of OPCs expressing high levels of transcription factors, such as Olig2, Nkx2.2 or Olig1 have been described during remyelination (Arnett et al., 2004; Fancy et al., 2004; Watanabe et al., 2004). Our results indicate that in human demyelinating diseases, similar to the findings in remyelinating animal models, OPCs react to a demyelinating stimulus.
The increased numbers of progenitors expressing high levels of Olig2 in early multiple sclerosis lesions may either be caused by the OPC migration, recruitment from a more immature cell population or proliferation. To analyse whether Olig2-positive cells proliferate in early disease stages, we performed double immunohistochemical staining with the proliferation marker Ki67 in two multiple sclerosis cases with high numbers of Olig2strong cells. In our study we rarely observed proliferating Olig2-positive cells. This is in line with an earlier publication demonstrating that the vast majority of proliferating cells in multiple sclerosis lesions are astrocytes and microglial cells (Schönrock et al., 1998) and may indicate a fundamental difference in the proliferating capacity of OPCs in human and mouse CNS (Levine and Reynolds, 1999).
In early multiple sclerosis lesions, we observed significantly lower numbers of Olig2strong cells in actively demyelinating as well demyelinated lesions compared with PPWM suggesting that either increased cell death or impaired migration of progenitors is the cause for low numbers of progenitors in these lesion areas.
In chronic lesions, we found a significantly reduced number of progenitors in PPWM and a trend to lower numbers of progenitors in demyelinated lesion areas compared with tissue specimens derived from patients with early multiple sclerosis confirming earlier studies (Wolswijk, 2000). However, in 8 out of 10 chronic demyelinated lesions Olig2strong-positive progenitors were still present. These results indicate that the limited remyelination found in chronic multiple sclerosis lesions is not simply caused by a lack of progenitors.
In addition to mature oligodendrocytes and progenitors, we found a subpopulation of oligodendroglial lineage cells co-expressing markers of mature oligodendrocytes (Nogo-A) as well as progenitors (high levels of Olig2) suggesting that these cells are either oligodendroglial cells in transition from OPCs to mature oligodendrocytes or mature oligodendrocytes recently recruited from the progenitor pool. This conclusion is supported by findings from a lysophatidylcholine induced demyelination model in which remyelination was associated with the presence of maturing oligodendrocytes expressing high levels of oligodendroglial transcription factors, such as Nkx2.2 (Watanabe et al., 2004). Together these findings suggest progenitors are present in early multiple sclerosis lesions and have the ability to develop into mature oligodendrocytes. In contrast, despite the presence of progenitor cells in chronic multiple sclerosis lesions, we observed few oligodendroglial lineage cells in transition to mature oligodendrocytes in chronic multiple sclerosis lesions suggesting a reduction in the differentiation capability of progenitors over time. There are numerous possible explanations for such a differentiation block in chronic multiple sclerosis lesions. One simple explanation could be the lack of contact between progenitors and axons. However, similar to an earlier study (Chang et al., 2002), we found progenitors closely associated with axons indicating that axons are either not receptive to remyelination or that inhibitory factors might prevent it. In recent studies, it has been shown that factors such as Lingo-1 and PSA-NCAM have the capability to prevent remyelination (Charles et al., 2000, 2002; Mi et al., 2005). However, the lack of certain factors such as IGF1, TGF-β1, GGF2 or integrins might also contribute to limited remyelination in multiple sclerosis lesions (Carson et al., 1993; McKinnon et al., 1993; Blaschuk et al., 2000; Mason et al., 2000). In toxin induced demyelinating animal models, depletion of inflammatory cells or lack of pro-inflammatory cytokines (e.g. IL1β, TNF) results in impaired remyelination indicating that the inflammatory infiltrates create a beneficial environment for remyelination (Arnett et al., 2001; Mason et al., 2001; Bieber et al., 2003; Kotter et al., 2005; Chari et al., 2006; Setzu et al., 2006). One major difference between early and chronic multiple sclerosis lesions is the markedly reduced inflammatory infiltrates suggesting that the altered inflammatory response in chronic multiple sclerosis lesions might contribute to the differentiation block of OPCs and limited extent of remyelination. Further studies are needed to identify the mechanisms responsible for the differentiation block of progenitors in multiple sclerosis.
In summary, we show that two types of Olig2- and Nkx2.2-positive cells are present in non-demyelinating CNS. Mature oligodendrocytes show a weak nuclear Olig2 or Nkx2.2 staining signal while progenitors express high levels of Olig2 and/or Nkx2.2. In early multiple sclerosis lesions, an increased number of progenitors and progenitors in transition to mature oligodendrocytes were observed compared with white matter from controls. In chronic multiple sclerosis lesions progenitors are still present but do not mature to oligodendrocytes. Our data support the hypothesis that a differentiation block of progenitors contributes to failed remyelination in multiple sclerosis. The development of neuroprotective treatments preventing the loss of progenitors and promoting the proliferation and differentiation of this cell population might be a promising treatment strategy for multiple sclerosis.
|
Acknowledgements |
|---|
We are grateful to Dr M.E. Schwab (Brain Research Institute, University of Zürich and Department of Biology, Swiss Federal Institute of Technology Zürich, Switzerland) for kindly providing the Nogo-A antibody 11C7. The authors wish to thank the Developmental Studies Hybridoma Bank (investigators: TM Jessell) for providing the Nkx2.2 antibody (developed under the auspieces of the NICHD and maintained by the University of Iowa, Departement of Biological Sciences, Iowa city, IA 52242). This study was supported by the Research Program and the Heidenreich von Siebold-Program from the Faculty of Medicine, Georg-August-University Göttingen, the Hertie Foundation and 6th Framework of the European Union, NeuroproMiSe, LSHM-CT-2005-018637 and the Foundation, Multiple Sclerosis Society of Canada.
|
References |
|---|
|
TopSummaryIntroductionMaterial and MethodsResultsDiscussionReferences |
|---|
Allen IV. Pathology of multiple sclerosis. In: McAlpine's multiple sclerosis—Matthews WB, ed. (1991) Edinburgh: Churchill Livingstone. 341–78.
Arnett HA, Fancy SP, Alberta JA, Zhao C, Plant SR, Kaing S, et al. bHLH transcription factor Olig1 is required to repair demyelinated lesions in the CNS. Science (2004) 306:2111–5.
Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JPY. TNF promotes proliferation of oligodendrocyte progenitors and remyelination. Nature Neurosci (2001) 4:1116–22.[CrossRef][Web of Science][Medline]
Barkhof F, Bruck W, De Groot CJ, Bergers E, Hulshof S, Geurts J, et al. Remyelinated lesions in multiple sclerosis: magnetic resonance image appearance. Arch Neurol (2003) 60:1073–81.
Barnett MH, Prineas JW. Relapsing and remitting multiple sclerosis: pathology of the newly forming lesion. Ann Neurol (2004) 55:458–68.[CrossRef][Web of Science][Medline]
Bieber AJ, Kerr S, Rodriguez M. Efficient central nervous system remyelination requires T cells. Ann Neurol (2003) 53:680–4.[CrossRef][Web of Science][Medline]
Blaschuk KL, Frost EE, Ffrench-Constant C. The regulation of proliferation and differentiation in oligodendrocyte progenitor cells by alphaV integrins. Development (2000) 127:1961–9.[Abstract]
Brück W, Porada P, Poser S, Rieckmann P, Hanefeld F, Kretzschmar HA, et al. Monocyte/macrophage differentiation in early multiple sclerosis lesions. Ann Neurol (1995) 38:788–96.[CrossRef][Web of Science][Medline]
Brück W, Schmied M, Suchanek G, Brück Y, Breitschopf H, Poser S, et al. Oligodendrocytes in the early course of multiple sclerosis. Ann Neurol (1994) 35:65–73.[CrossRef][Web of Science][Medline]
Butt A, Kiff J, Hubbard P, Berry M. Synantocytes: new functions for novel NG2 expressing glia. J Neurocytol (2002) 31:551–65.[CrossRef][Web of Science][Medline]
Carson MJ, Behringer RR, Brinster RL, McMorris FA. Insulin-like growth factor I increases brain growth and central nervous system myelination in transgenic mice. Neuron (1993) 10:729–40.[CrossRef][Web of Science][Medline]
Chang A, Tourtellotte WW, Rudick R, Trapp BD. Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. New Engl J Med (2002) 346:165–73.
Chari DM, Zhao C, Kotter MR, Blakemore WF, Franklin RJ. Corticosteroids delay remyelination of experimental demyelination in the rodent central nervous system. J Neurosci Res (2006) 83:594–605.[CrossRef][Web of Science][Medline]
Charles P, Hernandez MP, Stankoff B, Aigrot MS, Colin C, Rougon G, et al. Negative regulation of central nervous system myelination by polysialylated-neural cell adhesion molecule. Proc Natl Acad Sci USA (2000) 97:7585–90.
Charles P, Reynolds R, Seilhean D, Rougon G, Aigrot MS, Niezgoda A, et al. Re-expression of PSA-NCAM by demyelinated axons: an inhibitor of remyelination in multiple sclerosis? Brain (2002) 125:1972–9.
Copray S, Balasubramaniyan V, Levenga J, de Bruijn J, Liem R, Boddeke E. Olig2 overexpression induces the in vitro differentiation of neural stem cells into mature oligodendrocytes. Stem cells (2006) 24:1001–10.[CrossRef][Web of Science][Medline]
D'Souza SD, Alinauskas KA, Antel JP. Ciliary neurotrophic factor selectively protects human oligodendrocytes from tumor necrosis factor-mediated injury. J Neurosci Res (1996) 43:289–98.[CrossRef][Web of Science][Medline]
Fancy S, Zhao C, Franklin RJM. Increased expression of Nkx2.2 and Olig2 identifies reactive oligodendrocyte progenitor cells responding to demyelination in the adult CNS. Mol Cell Neurosci (2004) 27:247–54.[Web of Science][Medline]
Keirstead HS, Blakemore WF. Identification of post-mitotic oligodendrocytes incapable of remyelination within the demyelinated adult spinal cord. J Neuropathol Exp Neurol (1997) 56:1191–201.[Web of Science][Medline]
Kitada M, Rowitch D. Transcription factor co-expression patterns indicate heterogeneity of oligodendroglial subpopulations in adult spinal cord. Glia (2006) 54:35–46.[CrossRef][Web of Science][Medline]
Kotter MR, Zhao C, van RN, Franklin RJ. Macrophage-depletion induced impairment of experimental CNS remyelination is associated with a reduced oligodendrocyte progenitor cell response and altered growth factor expression. Neurobiol Dis (2005) 18:166–75.[CrossRef][Web of Science][Medline]
Kuhlmann T, Remington L, Maruschak B, Owens T, Bruck W. Nogo-A is a reliable oligodendroglial marker in adult human and mouse CNS and in demyelinated lesions. J Neuropathol Exp Neurol (2007) 66:238–46.[Web of Science][Medline]
Lassmann H, Raine CS, Antel J, Prineas JW. Immunopathology of multiple sclerosis: report on an international meeting held at the Institute of Neurology of the University of Vienna. J Neuroimmunol (1998) 86:213–7.[CrossRef][Web of Science][Medline]
Levine JM, Reynolds R. Activation and proliferation of rat endogenous oligodendrocyte precursor cells during ethidium bromide-induced demyelination. Exp Neurol (1999) 160:333–47.[CrossRef][Web of Science][Medline]
Ligon KL, Kesari S, Kitada M, Sun T, Arnett HA, Alberta JA, et al. Development of NG2 neural progenitor cells requires Olig gene function. Proc Natl Acad Sci (2006) 103:7853–8.
Lu Q, Sun T, Zhu Z, Ma N, Garcia M, Stiles CD, et al. Common developmental requirement for Olig2 function indicates a motor neuron/oligodendrocyte connection. Cell (2002) 109:75–86.[CrossRef][Web of Science][Medline]
Lucchinetti C, Brück W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. A quantitative analysis of oligodendrocytes in multiple sclerosis lesions. A study of 113 cases. Brain (1999) 122:2279–95.
Mason JL, Jones JJ, Taniike M, Morell P, Suzuki K, Matsushima GK. Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination. J Neurosci Res (2000) 61:251–62.[CrossRef][Web of Science][Medline]
Mason JL, Suzuki K, Chaplin DD, Matsushima GK. Interleukin-1beta promotes repair of the CNS. J Neurosci (2001) 21:7046–52.
Mason JL, Toews A, Hostettler JD, Morell P, Suzuki K, Goldmann JE, et al. Oligodendrocytes and progenitors become progressively depleted within chronically demyelinated lesions. Am J Pathol (2004) 164:1673–82.
McKinnon RD, Piras G, Ida JA Jr, Dubois-Dalcq M. A role for TGF-beta in oligodendrocyte differentiation. J Cell Biol (1993) 121:1397–407.
Mi S, Miller RH, Lee X, Scott ML, Shulag-Morskaya S, Shao Z, et al. LINGO-1 negatively regulates myelination by oligodendrocytes. Nat Neurosci (2005) 8:745–51.[CrossRef][Web of Science][Medline]
Miron VE, Jung CG, Kim HJ, Kennedy TE, Soliven B, Antel JP. FTY720 modulates human oligodendrocyte progenitor process extension and survival. Ann Neurol (2008) 63:61–71.[CrossRef][Web of Science][Medline]
Miron VE, Rajasekharan S, Jarjour AA, Zamvil SS, Kennedy TE, Antel JP. Simvastatin regulates oligodendroglial process dynamics and survival. Glia (2007) 55:130–43.[CrossRef][Web of Science][Medline]
Ozawa K, Suchanek G, Breitschopf H, et al. Patterns of oligodendroglia pathology in multiple sclerosis. Brain (1994) 117:1311–22.
Patani R, Balaratnam M, Vora A, Reynolds R. Remyelination can be extensive in multiple sclerosis despite a long disease course. Neuropathol Appl Neurobiol (2007) 33:277–87.[CrossRef][Web of Science][Medline]
Patrikios P, Stadelmann C, Kutzelnigg A, Rauschka H, Schmidbauer M, Laursen H, et al. Remyelination is extensive in a subset of multiple sclerosis patients. Brain (2006) 129:3165–72.
Penderis J, Shields SA, Franklin RJ. Impaired remyelination and depletion of oligodendrocyte progenitors does not occur following repeated episodes of focal demyelination in the rat central nervous system. Brain (2003) 126:1382–91.
Prineas JW. The neuropathology of multiple sclerosis. In: Demyelinating diseases—Koetsier JC, ed. (1985) Amsterdam: Elsevier Science Publishers. 213–57.
Prineas JW, Barnard RO, Kwon EE, Sharer LR, Cho ES. Multiple Sclerosis: remyelination of nascent lesions. Ann Neurol (1993) 33:137–51.[CrossRef][Web of Science][Medline]
Prineas JW, Connell F. Remyelination in multiple sclerosis. Ann Neurol (1979) 5:22–31.[CrossRef][Web of Science][Medline]
Qi Y, Cai J, Wu Y, Wu R, Lee J, Fu H, et al. Control of oligodendrocyte differentiation by the Nkx2.2 homeodomain transcription factor. Development (2001) 128:2723–33.
Raine CS, Scheinberg L, Waltz JM. Multiple sclerosis. Oligodendrocyte survival and proliferation in an active established lesion. Lab Invest (1981) 45:534–46.[Web of Science][Medline]
Raine CS, Wu E. Multiple sclerosis: remyelination in acute lesions. J Neuropathol Exp Neurol (1993) 52:199–204.[Web of Science][Medline]
Reynolds R, Dawson M, Papadopoulos D, Polito A, Di Bello IC, Pham-Dinh D, et al. The response of NG2-expressing oligodednrocyte progenitors to demyelination in MOG-EAE and MS. J Neurocytol (2002) 31:523–36.[CrossRef][Web of Science][Medline]
Ruffini F, Arbour N, Blain M, Olivier A, Antel JP. Distinctive properties of human adult brain-derived myelin progenitor cells. Am J Pathol (2004) 165:2167–75.
Schönrock ML, Kuhlmann T, Adler S, Bitsch A, Brück W. Identification of glial cell proliferation in early multiple sclerosis lesions. Neuropathol Appl Neurobiol (1998) 24:320–30.[CrossRef][Web of Science][Medline]
Setzu A, Lathia JD, Zhao C, Wells K, Rao MS, Ffrench-Constant C, et al. Inflammation stimulates myelination by transplanted oligodendrocyte precursor cells. Glia (2006) 54:297–303.[CrossRef][Web of Science][Medline]
Targett M, Sussman J, Scolding N, O’Leary MT, Compston DA, Blakemore WF. Failure to achieve remyelination of demyelinated rat axons following transplantation of glial cells obtained from the adult human brain. Neuropathol Appl Neurobiol (1996) 22:199–206.[CrossRef][Web of Science][Medline]
Watanabe M, Hadzic T, Nishiyama A. Transient upregulation of Nkx2.2 expression in oligodendrocyte lineage cells during remyelination. Glia (2004) 46:311–22.[CrossRef][Web of Science][Medline]
Wilson H, Scolding N, Raine C. Co-expression of PDGF receptor and NG2 by oligodendrocyte precursor cells in human CNS and multiple sclerosis lesions. J Neuroimmunol (2006) 176:162–73.[CrossRef][Web of Science][Medline]
Wolswijk G. Oligodendrocyte survival, loss and birth in lesions of chronic-stage multiple sclerosis. Brain (2000) 123:105–15.
Wolswijk G. Oligodendrocyte precursor cells in the demyelinated spinal cord. Brain (2002) 125:338–49.
Wosik K, Ruffini F, Almazan G, Olivier A, Nalbantoglu J, Antel JP. Resistance of human adult oligodendrocytes to AMPA/kainate receptor-mediated glutamate injury. Brain (2004) 127:2636–48.
Yong V, Antel J. Culture of glial cells from human brain biopsies. In: Protocols for neural cell culture—Fedoroff S, Richardson A, eds. (1992) New Jersey: Clifton. 76–81.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. P.J. Fancy, S. E. Baranzini, C. Zhao, D.-I. Yuk, K.-A. Irvine, S. Kaing, N. Sanai, R. J.M. Franklin, and D. H. Rowitch Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS Genes & Dev., July 1, 2009; 23(13): 1571 - 1585. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Kremer, A. Heinen, J. Jadasz, P. Gottle, K. Zimmermann, P. Zickler, S. Jander, H.-P. Hartung, and P. Kury p57kip2 is dynamically regulated in experimental autoimmune encephalomyelitis and interferes with oligodendroglial maturation PNAS, June 2, 2009; 106(22): 9087 - 9092. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Goldschmidt, J. Antel, F. B. Konig, W. Bruck, and T. Kuhlmann Remyelination capacity of the MS brain decreases with disease chronicity Neurology, June 2, 2009; 72(22): 1914 - 1921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Melani, S. Cipriani, M. G. Vannucchi, D. Nosi, C. Donati, P. Bruni, M. G. Giovannini, and F. Pedata Selective adenosine A2a receptor antagonism reduces JNK activation in oligodendrocytes after cerebral ischaemia Brain, June 1, 2009; 132(6): 1480 - 1495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. E. Miron, S. P. Zehntner, T. Kuhlmann, S. K. Ludwin, T. Owens, T. E. Kennedy, B. J. Bedell, and J. P. Antel Statin Therapy Inhibits Remyelination in the Central Nervous System Am. J. Pathol., May 1, 2009; 174(5): 1880 - 1890. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Pescini Gobert, L. Joubert, M.-L. Curchod, C. Salvat, I. Foucault, C. Jorand-Lebrun, M. Lamarine, H. Peixoto, C. Vignaud, C. Fremaux, et al. Convergent Functional Genomics of Oligodendrocyte Differentiation Identifies Multiple Autoinhibitory Signaling Circuits Mol. Cell. Biol., March 15, 2009; 29(6): 1538 - 1553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Baer, Y. A. Syed, S. U. Kang, D. Mitteregger, R. Vig, C. ffrench-Constant, R. J. M. Franklin, F. Altmann, G. Lubec, and M. R. Kotter Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling Brain, February 1, 2009; 132(2): 465 - 481. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Buckley, P. Goldsmith, and R. J. M. Franklin Zebrafish myelination: a transparent model for remyelination? Dis. Model. Mech., November 1, 2008; 1(4-5): 221 - 228. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||