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Astrocyte Activation via Stat3 Signaling Determines the Balance of Oligodendrocyte versus Schwann Cell Remyelination
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The American Journal of Pathology, Vol. -, No. -, - 2015
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Astrocyte Activation via Stat3 Signaling Determines the Balance of Oligodendrocyte versus Schwann Cell Remyelination Q35
Glaucia Monteiro de Castro,*y Natalia A. Deja,* Dan Ma,* Chao Zhao,* and Robin J.M. Franklin* From the Wellcome Trust-MRC Cambridge Stem Cell Institute, Department of Clinical Neurosciences,* University of Cambridge, Cambridge, United Kingdom; and the Department of Biosciences,y Sao Paulo Federal University, Santos, Brazil Accepted for publication May 7, 2015.
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Address correspondence to Robin J.M. Franklin or Chao Zhao, Wellcome Trust-MRC Cambridge Stem Cell Institute and Department of Clinical Neurosciences, Clifford Allbutt Building, Cambridge Biomedical Campus, University of Cambridge, Cambridge, United Kingdom. E-mail: rjf1000@ cam.ac.uk or [email protected]. uk.
Remyelination within the central nervous system (CNS) most often is the result of oligodendrocyte progenitor cells differentiating into myelin-forming oligodendrocytes. In some cases, however, Schwann cells, the peripheral nervous system myelinating glia, are found remyelinating demyelinated regions of the CNS. The reason for this peripheral type of remyelination in the CNS and what governs it is unknown. Here, we used a conditional astrocytic phosphorylated signal transducer and activator of transcription 3 knockout mouse model to investigate the effect of abrogating astrocyte activation on remyelination after lysolecithin-induced demyelination of spinal cord white matter. We show that oligodendrocyte-mediated remyelination decreases and Schwann cell remyelination increases in lesioned knockout mice in comparison with lesioned controls. Our study shows that astrocyte activation plays a crucial role in the balance between Schwann cell and oligodendrocyte remyelination in the CNS, and provides further insight into remyelination of CNS axons by Schwann cells. (Am J Pathol 2015, -: 1e10; http://dx.doi.org/ 10.1016/j.ajpath.2015.05.011)
The adult mammalian central nervous system (CNS) is remarkably efficient at replacing myelin-forming cells after primary demyelination.1 This regenerative process is called remyelination. Although in most circumstances the new myelin-forming cells are oligodendrocytes, the myelinating cells of the CNS, it also has been well established in both experimental models and clinical disease that remyelination can be mediated by Schwann cells, the myelinating cells of the peripheral nervous system.2e8 New remyelinating oligodendrocytes are generated from a population of neural progenitor cells that are distributed widely throughout the adult CNS.9e11 These cells can be identified using a range of markers, of which two commonly used markers are Pdgfra and NG2, and generally are referred to as oligodendrocyte progenitor cells (OPCs).12 In the past, it was assumed that all Schwann cells engaged in remyelinating CNS axons were derived from peripheral nervous system Schwann cells, and entered the CNS through breaches in the astrocytic glia limitans.13,14 Although this is certainly a source of some CNS Schwann cells,10 transplantation15 and
genetic fate mapping studies10 have shown that large numbers of CNS Schwann cells are derived from OPCs. What remains unclear is how and why Schwann cell remyelination occurs within the CNS. Clues are provided by the anatomic features associated with areas of the CNS in which either oligodendrocyte or Schwann cell remyelination occurs. The most consistently observed feature is the astrocyte status: oligodendrocyte remyelination occurs in regions where astrocytes are present, restoring a complete CNS glial environment, whereas Schwann cell remyelination occurs where astrocytes are absent, resulting in patches of tissue that resemble the peripheral nervous system.16e19 Indeed, the extent of Schwann cell remyelination is directly proportional to the extent of astrocyte absence. This is seen most clearly when comparing the remyelination of ethidium bromideeinduced Supported by grants from the UK Multiple Sclerosis Society, CNPq 200993/2010-0 (G.M.d.C.) and 201797/2011-9, and Ciência Sem Fronteiras. Disclosures: None declared.
Copyright ª 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2015.05.011
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spinal cord demyelination, in which there initially is extensive astrocyte loss and hence a high proportion of Schwann cell remyelination, with remyelination of lysolecithin-induced spinal cord demyelination, which is more sparing of astrocytes and hence has a smaller Schwann cell contribution to remyelination.20 The extent of Schwann cell remyelination has been manipulated experimentally using cell transplantation approaches, in which, for example, the extent of Schwann cell remyelination of ethidium bromideeinduced spinal cord demyelination can be reduced by astrocyte transplantation.21,22 However, whether astrocyte responses play a physiological role in determining the balance of central versus peripheral types of remyelination has not been tested in experiments in which the endogenous astrocyte response is altered. In this study we took advantage of the central role of phosphorylation of the transcription factor signal transducer and activator of transcription 3 (Stat3) in the astrocyte response to CNS injury23 to address the hypothesis that the proportion of Schwann cell remyelination after experimental demyelination is dependent on astrocyte activation within the lesion. By using a conditional Cre-loxP approach we were able to specifically prevent Stat3 phosphorylation in astrocytes after focal toxin-induced demyelination. We showed that not only did this lead to a reduced astrocyte response, it also led to an impairment in OPC activation and resulted in an increased level of Schwann cell remyelination and a decreased level of oligodendrocyte remyelination, thereby showing a central role of astrocyte activation in determining the nature of CNS remyelination.
Materials and Methods Animals
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The astrocyte-specific phosphorylated Stat3 (pStat3) conditional knockout mouse strain [glial fibrillary acidic protein (GFAP)-STAT3-CKO] on a C57BL6 background kindly was provided by Dr. Michael Sofroniew (University of California, Los Angeles, CA).23 Ablation of the activated form of Stat3 in astrocytes was achieved by conditional Cre-loxP recombination. In this strain, the Cre recombinase is expressed under the mouse GFAP promoter (GFAP-Cre). Recombination occurs at loxP sites flanking exon 22 (the phosphorylation site containing exon) of the Stat3 gene (Stat3fl/fl). As a result, phosphorylation of Stat3, crucial for its function, does not occur. Experimental animals were bred using homozygous Stat3fl/fl males and heterozygous Cre-expressing females (GFAP-Creþ/-:Statfl/fl). The resulting GFAP-STAT3-CKO mice showed normal development and were fertile. Both male and female mice were used in the experiments, with noneCre-expressing littermates (Statfl/fl) used as controls. Experiments were performed in compliance with UK Home Office regulations and institutional guidelines.
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Toxin-Induced Demyelination Focal spinal cord demyelination was created as previously described.24 Briefly, 8- to 10-week-old mice were anesthetized with isoflurane and the spinal cord was exposed at the level of T12/T13 by removing the soft tissue between the vertebrae. One microliter of 1% L-a-lysophosphatidylcholine (lysolecithin; Sigma-Aldrich, Gilingham, UK) in sterile saline was injected using a Hamilton syringe fitted Q9 with a fine glass tip into the ventral spinal cord white matter (Figure 1A). At designated time points after injection, mice ½F1 were terminally anesthetized with an overdose of pentobarbital before being perfused transcardially with either 4% paraformaldehyde for immunohistochemistry or 4% glutaraldehyde for resin embedding and electron microscopy.
Immunohistochemistry Immunohistochemistry was performed on 12-mmethick frozen sections. When required, heat-mediated antigen retrieval with 10 mmol/L sodium citrate buffer (pH 6) was performed before the standard protocol for indirect immunofluorescence staining. Briefly, slide-mounted sections were washed with phosphate-buffered saline (pH 7.4), and blocked with 5% normal donkey serum and 0.1% Triton X-100 in phosphate-buffered saline for 1 hour at Q10 room temperature. Sections then were incubated with primary antibodies diluted in blocking solution overnight at 4 C. The following primary antibodies were used: Olig2 1:200 (Millipore, Watford, UK), CC1 1:100 (Apc; Cal- Q11 biochem, San Diego, CA), ionized calcium binding adaptor molecule 1 (Iba1) 1:500 (Wako, Osaka, Japan), pStat3 (Tyr705) 1:100 (Cell Signaling Technology, Beverly, MA), and Aldh1l1 1:100 (clone N103/39; Neuromab, Davis, CA). For Apc and Aldh1l1 staining on lesion sections, Mouse On Mouse blocking reagents (Vector Labs) Q12 Q13 were used to reduce nonspecific background according to the manufacturer’s instructions. Staining was visualized with Alexa Fluoreconjugated secondary antibodies (1:500; Invitrogen, Paisley, UK). The slides were counterstained with 0.1% Sudan Black (Sigma-Aldrich) to reduce background and show the lesion area, and subsequently were mounted in FluorSave Reagent (Calbiochem). The images were acquired using a Zeiss Axio Observer fluorescence microscope.
In Situ Hybridization In situ hybridization of digoxigenin-labeled complementary RNA probes for myelin protein zero and myelin proteolipid protein was performed as previously described.25 Briefly, Q14 sections were hybridized with digoxigenin-labeled complementary RNA probes at 65 C overnight and subjected to a standard wash protocol (50% formamide, 1 standard saline citrate, 0.1% Tween-20, 65 C, 3 30 minutes) to remove nonspecific probe binding. The target bound probes were
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Figure 1 Expression of phosphorylated signal transducer and activator of transcription 3 (pStat3) after focal demyelination in spinal cord white matter is reduced in glial fibrillary acidic protein (GFAP) promoter-controlled conditional Stat3 knockout mice. A: Control and mutant animals were injected with lysolecithin to create a focal demyelination lesion. B and C: The quantification of the total numbers of pStat3þ (B) and pStat3/Gfap double-positive (C) cells in control and GFAP-STAT3-CKO lesions. DeI: Immunostaining of lesioned ventral spinal cord white matter at 14 days after lesion (dpl). D and G: Merged immunostaining for phosphorylated Stat3 (pStat3), and the nuclear dye Hoechst 33342. The dotted line marks the border of the demyelinated area, as demarcated by increased cellularity shown by Hoechst staining (inset in D). E and H: Co-labeling with pStat3 and astrocyte marker Gfap; boxed area in each image is magnified in panels F and I, respectively. J and K: Representative images show colocalization of an alternative astrocyte marker Aldh1l1 and pStat3 in demyelinated areas at 14 dpl, in control (J) and GFAP-STAT3-CKO (K) animals. LeO: Quantification is shown. pSTAT3 also is expressed in other types of cells, including CD11bþ macrophages/ microglia (M and N) and Olig2þ oligodendrocyte lineage cells (O) within lesions. Means SEM are shown. Arrowheads (F, J, K, and MeO) indicate cells labeled with both markers. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (DeI); 25 mm (J and K). WT, wild type.
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embedding.24 Lesions were localized on semithin 1-mm sections stained with toluidine blue. Ultrathin sections of the lesion site were cut onto copper grids and stained with uranyl acetate before being examined with a Hitachi Q15 H-600 Transmission Electron Microscope.
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For each animal, three demyelinated lesion sections, separated by approximately 120 mm, were selected from within the central region of the lesion. For immunostaining, the outline of each lesion was defined based on the increase in cellularity inside the lesion, as visualized by Hoechst 33342 counterstain. For in situ hybridization, the outline was defined based on the lesioned tissue texture, using Zeiss AxioVision software. The number of Q16 marker-positive cells inside the lesions was counted manually using ImageJ version x.xxx (NIH, Bethesda, Q17 MD; http://imagej.nih.gov/ij), and normalized against the lesion area. The average of three sections was used for each lesioned animal. For each group, four to five animals were used. To compare differences between the control and experimental groups, a two-way analysis of variance followed by the Bonferroni post-test was used, and the threshold for statistical significance was set at P < 0.05.
Figure 2
Electron Microscopy Animals were perfused with 4% glutaraldehyde in phosphate-buffered saline containing 0.4 mmol/L CaCl2. The spinal cord was sliced coronally at 1-mm thickness and treated with 2% osmium tetroxide overnight before being subjected to a standard protocol for epoxy resin
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Figure 3 Macrophage and microglia infiltration after demyelination in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO mice is similar to that in controls. The microglia/macrophages in the demyelinated area at 5 days after lesion (dpl) were visualized with antibody against macrophage/microglia marker ionized calcium binding adaptor molecule 1 (Iba1) in control (A) and GFAPSTAT3-CKO (B) mice. C: Mean optical density was used to compare the Iba1 immunoreactivity between controls and mutants across time points. D: The average macrophage/microglia infiltration area at different survival times. Measurements were taken from transverse sections of the lesion that had the largest lesion area (identified by increased cellularity). Means SEM. Scale bars: 50 mm (A and B).
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detected by alkaline phosphataseeconjugated antidigoxigenin antibody, and visualized as purple precipitate after incubation in NBT/BCIP solution according to the manufacturer’s instructions (Roche, Lewes, UK). The slides were dehydrated with ascending concentration of ethanol, cleared with xylene, and mounted in dibutyl phthalate in xylene. Images were acquired with the Zeiss Axio Observer microscope.
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Astrocyte response to demyelination is attenuated in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO mice. AeD: Images illustrate areas of lysolecithininduced demyelination in ventral spinal cord white matter at 14 days after lesion (dpl), immunolabeled for astrocyte marker Gfap and nucleus dye Hoechst 33342 (A and C), with lesion areas demarcated by counterstaining with Sudan Black (B and D). Graphs show the relative total area occupied by immunoreactive EeG: Gfap staining in demyelinated lesions (E) and the average length of astrocytic processes in control and GFAP-STAT3-CKO mice (F), and the number of astrocytes inside the lesion area identified by the astrocyte marker Aldh1l1 (G). Dotted lines mark the lesion boundaries. Means SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (AeD).
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The response of oligodendrocyte lineage cells to lysolecithininduced spinal cord demyelination in glial fibrillary acidic protein (GFAP)signal transducer and activator of transcription 3 (STAT3)-CKO mice. AeD: Demyelinated areas of ventral spinal cord lesions at 14 days after lesion (dpl) from control and GFAP-pSTAT3-CKO mice double-stained for oligodendrocyte lineage marker Olig2 and transcription factor Sox2, a marker for activated oligodendrocyte progenitor cells. B and D: Enlarged areas marked by an orange rectangle in panels A and C, respectively. EeG: Densities of single-labeled and co-labeled cells are compared. Arrows indicate double-labeled cells. Means SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (A and B).
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559 560 561 Expression of Phosphorylated Stat3 in Astrocytes Is 562 Reduced in GFAP-STAT3-CKO Mice after Toxin-Induced 563 CNS Demyelination 564 565 Phosphorylation of Stat3 is increased in multiple CNS cell 566 25e27 types during injury. To verify this in our demyelination 567 model we examined the expression of pStat3 in control, 568 noneCre-expressing lesioned and unlesioned mice, using a 569 pStat3-specific antibody. To obtain a focal demyelinating 570 lesion, animals were injected with 1% lysolecithin solution 571 572 into the ventral spinal cord (Figure 1A). In normal unle573 sioned spinal cord, very few cells were found to express 574 pStat3 (not shown). In contrast, there was a marked increase 575 in pStat3 expression 5 days after lesion (dpl) (Figure 1B). 576 The pStat3 levels remained increased at 14 and 21 dpl, 577 albeit they were somewhat decreased compared with the 578 expression at 5 dpl (Figure 1B). The staining was most 579 intense within the nucleus (Figure 1D). pStat3þ cells 580 included a variety of cell types, such as CD11bþ macro581 phages/microglia, Olig2þ oligodendrocyte lineage cells, and 582 Gfapþ astrocytes (Figure 1, E, F, MeO). 583 584 Stat3 phosphorylation plays a key role in mediating 585 astrocyte responses to CNS injury.23 To explore the 586 astrocyte-specific role of pStat3 in lysolecithin-induced 587 demyelination further, we used a conditional Stat3 588 knockout mouse model in which Cre recombinase was 589 expressed under the GFAP promoter. The Cre recombinase 590 excised the floxed Stat3 exon 22 containing the phosphor591 ylation site (Tyr 705) involved in Stat3 activation,23 592 resulting in a mutant unable to phosphorylate Stat3 in as593 trocytes (GFAP-STAT3-CKO mice). We first investigated 594 pStat3 expression in lesioned GFAP-STAT3-CKO mice. As 595 596 expected, the number pStat3-expressing astrocytes, as 597 examined by co-labeling with astrocyte markers Gfap 28 598 (Figure 1, CeI) and aldehyde dehydrogenase 1 (Aldh1l1) þ 599 surrounding pStat3 nuclei, was significantly lower in the 600 mutants compared with controls (Figure 1, JeL). 601 602 Demyelination-associated Astrogliosis Is Reduced in 603 604 GFAP-STAT3-CKO Mice 605 606 Lysolecithin demyelination is characterized by an abundance of astrocytes within the lesioned area (Figures 1, CeL, and 2, ½F2 607 608 AeD). In control mice, we observed an increase in Gfap 609 immunoreactivity within and beyond the demyelinated area 610 (the latter was determined by the lipophilic dye Sudan Black) 611 (Figure 2, A and B). An increase in Gfap expression also was 612 seen in demyelinated areas of the spinal cord in GFAP613 STAT3-CKO mice when compared with unlesioned tissue 614 surrounding the lesioned areas (Figures 1H, and 2, C and D), 615 although the intensity of the staining was lower than in 616 617 control animals (not shown). Because Gfap staining localizes 618 to astrocytic processes that often form a tangled mesh in 619 injured tissue, rendering quantification of individual positive 620
Results
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621 Control 21DPL B STAT3CKO 21DPL C CC1+ cells A 622 1200 623 1000 624 800 625 600 626 400 ** 627 * 200 628 0 629 14 DPL 21 DPL CC1Hoechst CC1Hoechst 630 631 plp+ cells Control 21DPL E STAT3CKO 21DPL F 632 D 1000 633 634 800 635 600 636 400 * ** 637 200 638 639 0 plp plp 14 DPL 21 DPL 640 641 Figure 5 Oligodendrocyte remyelination is reduced in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO 642 mice. Remyelination by oligodendrocytes in control and GFAP-pSTAT3-CKO mice at 14 and 21 days after lesion (dpl) was assessed by immunostaining for the 643 differentiated oligodendrocyte marker CC1 (A and B) and in situ hybridization for proteolipid protein (plp) mRNA (D and E). Images show representative Q29 644 examples of demyelinated lesions at 21 dpl. C and F: The cell densities for each marker at selected survival times are compared. Means SEM are shown. Q30 *P < 0.01 and **P < 0.001. Scale bars: 100 mm (A, B, D, and E). 645 646 cells challenging, we instead compared the areas occupied by the conditional astrocytic pStat3 ablation on oligodendro647 reactive astrocytes in control and GFAP-STAT3-CKO spinal cyte remyelination by comparing the distribution of oligo648 649 cord lesions, as defined by a clear boundary of increased Gfap dendrocyte lineage cells using different markers expressed 650 reactivity. We found the relative area of reactive astrocytes at specific stages of lineage progression in control and 651 Q18 over total lesion area to be reduced in GFAP-STAT3-CKO mutant lesioned mice. In control animals, Olig2þ cell 652 mice at both 5 and 14 dpl (Figure 2E). This was verified by numbers increased at 5 dpl (when OPCs are recruited 653 counting the number of Aldh1l1þ cells in the demyelinated actively to the lesion), increased further at 14 dpl (when 654 differentiation is ongoing), and remained high at 21 dpl areas, which showed a significant 40% to 60% reduction in 655 (when remyelination is near completion) (Figure 4, A, B, ½F4 GFAP-STAT3-CKO mice compared with controls at all 656 Q19 three survival time points examined (Figure 2G and E). In GFAP-STAT3-CKO animals, the density of ). 657 þ Olig2 cells was comparable with controls at 5 dpl, but was 658 Reducing Stat3 Activation in Astrocytes Does Not Alter reduced significantly at 14 and 21 dpl (Figure 4, CeE). 659 Macrophage Responses to Demyelination Increased expression of the transcription factor Sox2 is a 660 661 marker of OPC activation (unpublished observations). The Abrogation of Stat3 activation results in spread of inflammation 662 density of total Sox2þ cells and Sox2þ/Olig2þ co-labeled after a spinal cord crush injury.23,29 To assess the influence of 663 cells in the demyelinated area were significantly lower in 664 astrocytic Stat3 knockout in our demyelination model, we the GFAP-STAT3-CKO group compared with controls at 5 665 examined the microglia/macrophage response in GFAPand 14 dpl, suggesting impaired OPC activation (Figure 4, F 666 STAT3-CKO mice after demyelination by immunostaining for and G). There was also a reduction in Sox2þ cells that did 667 Iba1. Iba1þ cells were present at high density throughout the not express Olig2, which are likely to be astrocytes, and this 668 ½F3 lesion (Figure 3). It was not feasible to quantify the microglia/ is consistent with the data in Figure 2G. The reduced density 669 macrophage cellular density because of the fused pattern of of Olig2þ and Olig2þ/Sox2þ cells was mirrored by a 670 immunostaining in lesions, therefore we measured the normalreduced expression of the mature oligodendrocyte markers 671 ized mean optical density to represent the extent of microglia/ CC1 and myelin proteolipid protein at 14 and 21 dpl 672 macrophage infiltration in the demyelinated area. There was no 673 (Figure 5). ½F5 difference in either Iba1 intensity or the area of Iba1þ cell 674 infiltration at all of the time points examined (Figure 3, C and D). 675 Attenuated Astrogliosis Increases Schwann Cell CNS 676 The Attenuated Astrocyte Response Reduces 677 Remyelination 678 Oligodendrocyte Remyelination 679 The mutual exclusivity of the presence of astrocytes and Q20 680 Schwann cell remyelination within the same region of repaired Astrocytes are known to influence OPCs during CNS 681 demyelinated lesions led us to reason that an attenuated remyelination.21,30e32 We therefore assessed the impact of 682
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Figure 6 Schwann cell remyelination increases in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO mice. Remyelinating Schwann cells from control and GFAP-STAT3-CKO mice at 21 days after lesion (dpl) were examined by periaxin immunostaining (AeD) and myelin protein zero (mpz) mRNA in situ hybridization (FeH). Dotted lines (AeD, F, and G) mark demyelinated areas. E and F: Quantification of the relative area of positive periaxin immunofluorescence (E) and cells containing mpz mRNA (F). J and K: Semithin resin sections from control (J) and STAT3CKO (K) lesioned mice were stained with toluidine blue. Areas of oligodendrocyte remyelination are marked with the yellow letter O, and that of Schwann cells are marked with the red letter S. M and N: Enlarged boxed areas from panels J and K, respectively. L and O: Examples of axons remyelinated by oligodendrocytes are marked by yellow arrowheads; green arrowheads point to examples of axons that were not demyelinated, blue arrowheads indicate poorly remyelinated axons, and red arrowheads mark typical morphology of myelinating Schwann cells. These observations were verified further by electron microscopy with examples shown in control (L) and GFAP-STAT3-CKO (O) cells. Inset (O) shows an enlarged view of the boxed area, showing the typical structure of myelinating Schwann cells. Means SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (AeD, F, G, JeM); 5 mm (N and O).
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Monteiro de Castro et al 869 astrocytic response could lead to increased Schwann cell 870 remyelination in GFAP-STAT3-CKO mutants. Indeed, 871 compared with lesioned controls, the GFAP-STAT3-CKO 872 spinal cord white matter lesions showed increased areas of 873 periaxin antigenicity and increased density of myelin protein 874 zero mRNA expression at 14 and 21 dpl, indicating increased 875 876 ½F6 Schwann cell remyelination (Figure 6, AeI). The increased number of remyelinating Schwann cells was confirmed by 877 analyzing semithin resin sections and performing electron 878 879 microscopy, where Schwann cells could be recognized readily 880 by their typical signet-ring morphology and relatively thicker 881 myelin sheath (Figure 6, JeO). Notably, in the GFAP-STAT3882 CKO lesions, Schwann cell remyelinated areas contained more 883 demyelinated axons than oligodendrocyte remyelinated areas at 884 all of the time points examined (Figure 6, M and N). Because 885 toxin-induced demyelination invariably undergoes complete 886 remyelination, it is likely that these few remaining demyelinated 887 axons eventually will undergo remyelination. 888 889 890 891 Discussion 892 893 In this study, we used a transgenic conditional astrocytic 894 pStat3 knockout model to show that altering astrocyte 895 activation significantly influences the response to CNS 896 demyelination injury. This model had been used previously 897 to study astrogliosis and scar formation in spinal cord 898 trauma.23 In accordance with this previous study, we found 899 that abrogating astrocyte activation decreased the astrocytic 900 response in the lesion. However, unlike in the trauma 901 model, we found no effect of pStat3 manipulation in as902 903 trocytes on macrophage responses.29 Crucially, our study 904 focused on remyelination and showed that oligodendrocyte 905 remyelination was reduced and Schwann cell remyelination 906 was increased in lesioned GFAP-STAT3-CKO mice. Our 907 findings therefore constitute the first direct proof of the 908 indispensable role of astrocyte activation in the balance 909 between Schwann cell and oligodendrocyte remyelination in 910 the CNS. 911 The intriguing phenomenon of Schwann cell remyelina912 tion within the CNS has been recognized for many decades, 913 however, its mechanisms and functions remain obscure. It 914 915 now is known, as a result of genetic fate mapping strategies, 916 that contrary to the previously held belief, many of the 917 Schwann cells that appear in the CNS are not immigrants 918 from the peripheral nervous system but instead are derived 919 from CNS progenitor cells.10 It also has been recognized 920 that Schwann cell remyelination often occurs around blood 921 vessels and within areas of damaged CNS from which 922 astrocytes are absent.16,19,33 This observation has led to the 923 hypothesis that the presence or absence of astrocytes de924 termines if CNS progenitor cells support remyelination of 925 demyelinated axons by becoming an oligodendrocyte or a 926 927 Schwann cell. Our results clearly show a central role for 928 astrocytes in determining the balance of central versus 929 peripheral-type remyelination. It remains unclear how 930
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astrocytes exert this effect. One proposed hypothesis is that members of the bone morphogenetic protein family induce OPCs to become Schwann cells, however, this is prevented in the presence of astrocytes by astrocyte-derived inhibitors of bone morphogenetic protein signaling, with OPCs becoming oligodendrocytes instead. However, although there is some evidence that the fate of transplanted OPCs can be influenced by prior treatment in vitro with bone morphogenetic proteins,34,35 there is no compelling evidence for such mechanisms in vivo. Because the current STAT3 knockout takes place at the first appearance of GFAP expression during development, it is possible that there are long-term changes in the environment that contribute to the shift in remyelination type. However, if Q21 such changes do exist they would seem to be shown only after injury because phenotypic changes have not been identified in the absence of injury in either our study or in previous studies on astrocyte STAT3-null animals. Thus, Q22 exactly how OPCs become Schwann cells in the CNS in the absence of astrocytes remains to be fully explored. What is the functional significance of Schwann cells myelinating CNS axons? There are two main functions of myelin: to allow rapid saltatory conduction and to help maintain axon health and integrity.36 It has been evident for many years that Schwann cell myelination restores saltatory conduction to demyelinated CNS axons, and from this perspective it appears to make no difference which type of myelin surrounds the axons.37,38 However, the relative effect of peripheral versus central-type myelin on axonal integrity is entirely unknown. Schwann cells and oligodendrocytes differ in a number of ways: they develop from different tissues, use different strategies to myelinate target axons, produce different extracellular components, and assemble molecularly distinct nodes and paranodes.39 Moreover, key differences have been shown in their metabolic relationships with the axons they ensheath.40 Therefore, it is possible that in the context of recovery from CNS demyelinating injury, Schwann cell CNS remyelination may have distinctive physiological advantages compared with oligodendrocyte remyelination, although this hypothesis remains to be tested. Certainly, in the context of immune-mediated damage directed against epitopes specific to oligodendrocytes and their myelin, one might imagine that the Schwann cell would be resistant to direct injury and therefore this form of remyelination might protect against subsequent oligodendrocyte-directed immune attack. Our study has shown that a substantial reduction of pStat3-mediated astrocyte activation is a sufficient prerequisite to sway the remyelination process toward peripheral nervous systemetype remyelination in the CNS. This evidence will help to elucidate further the development and function of peripheral-type CNS remyelination, which presents not only an intriguing biological phenomenon but also an interesting and unexplored therapeutic possibility.
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Acknowledgments We thank Dr. Michael Sofroniew for providing the GFAPSTAT3-CKO mice, and Michael Peacock and Daniel Morrison for their help with electron microscopy.
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stereotaxic injections of lysolecithin, ethidium bromide and complement/anti-galactocerebroside: a comparative study. Glia 1999, 25:216e228 Itoyama Y, Ohnishi A, Tateishi J, Kuroiwa Y, Webster HD: Spinal cord multiple sclerosis lesions in Japanese patients: Schwann cell remyelination occurs in areas that lack glial fibrillary acidic protein (GFAP). Acta Neuropathol 1985, 65:217e223 Talbott JF, Loy DN, Liu Y, Qiu MS, Bunge MB, Rao MS, Whittemore SR: Endogenous Nkx2.2(þ)/Olig2(þ) oligodendrocyte precursor cells fail to remyelinate the demyelinated adult rat spinal cord in the absence of astrocytes. Exp Neurol 2005, 192:11e24 Blakemore WF, Franklin RJM: Remyelination in experimental models of toxin-induced demyelination. Curr Top Microbiol Immunol 2008, 318:193e212 Franklin RJM, Crang AJ, Blakemore WF: Transplanted type-1 astrocytes facilitate repair of demyelinating lesions by host oligodendrocytes in adult rat spinal cord. J Neurocytol 1991, 20: 420e430 Blakemore WF, Gilson JM, Crang AJ: The presence of astrocytes in areas of demyelination influences remyelination following transplantation of oligodendrocyte progenitors. Exp Neurol 2003, 184: 955e963 Herrmann JE, Imura T, Song B, Qi J, Ao Y, Nguyen TK, Korsak RA, Takeda K, Akira S, Sofroniew MV: STAT3 is a critical regulator of astrogliosis and scar formation after spinal cord injury. J Neurosci 2008, 28:7231e7243 Zhao C, Fancy SPJ, Ffrench-Constant C, Franklin RJM: Osteopontin is extensively expressed by macrophages following CNS demyelination but has a redundant role in remyelination. Neurobiol Dis 2008, 31: 209e217 Sriram K, Benkovic SA, Hebert MA, Miller DB, O’Callaghan JP: Induction of gp130-related cytokines and activation of JAK2/STAT3 pathway in astrocytes precedes up-regulation of glial fibrillary acidic protein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of neurodegeneration: key signaling pathway for astrogliosis in vivo? J Biol Chem 2004, 279:19936e19947 Acarin L, Gonzalez B, Castellano B: STAT3 and NFkappaB activation precedes glial reactivity in the excitotoxically injured young cortex but not in the corresponding distal thalamic nuclei. J Neuropathol Exp Neurol 2000, 59:151e163 Yamauchi K, Osuka K, Takayasu M, Usuda N, Nakazawa A, Nakahara N, Yoshida M, Aoshima C, Hara M, Yoshida J: Activation of JAK/STAT signalling in neurons following spinal cord injury in mice. J Neurochem 2006, 96:1060e1070 Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA: A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 2008, 28:264e278 Faulkner JR, Herrmann JE, Woo MJ, Tansey KE, Doan NB, Sofroniew MV: Reactive astrocytes protect tissue and preserve function after spinal cord injury. J Neurosci 2004, 24:2143e2155 Williams A, Piaton G, Lubetzki C: Astrocytesefriends or foes in multiple sclerosis? Glia 2007, 55:1300e1312 Albrecht PJ, Murtie JC, Ness JK, Redwine JM, Enterline JR, Armstrong RC, Levison SW: Astrocytes produce CNTF during the remyelination phase of viral-induced spinal cord demyelination to stimulate FGF-2 production. Neurobiol Dis 2003, 13:89e101 Hammond TR, Gadea A, Dupree J, Kerninon C, Nait-Oumesmar B, Aguirre A, Gallo V: Astrocyte-derived endothelin-1 inhibits remyelination through notch activation. Neuron 2014, 81:588e602 Sim FJ, Zhao C, Li WW, Lakatos A, Franklin RJM: Expression of the POU domain transcription factors SCIP/Oct-6 and Brn-2 is associated with Schwann cell but not oligodendrocyte remyelination of the CNS. Mol Cell Neurosci 2002, 20:669e682 Talbott JF, Cao Q, Enzmann GU, Benton RL, Achim V, Cheng XX, Mills MD, Rao MS, Whittemore SR: Schwann cell-like
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Monteiro de Castro et al differentiation by adult oligodendrocyte precursor cells following engraftment into the demyelinated spinal cord is BMP-dependent. Glia 2006, 54:147e159 35. Crang AJ, Gilson JM, Li WW, Blakemore WF: The remyelinating potential and in vitro differentiation of MOG-expressing oligodendrocyte precursors isolated from the adult rat CNS. Eur J Neurosci 2004, 20:1445e1460 36. Nave KA, Werner HB: Myelination of the nervous system: mechanisms and functions. Annu Rev Cell Dev Biol 2014, 30:503e533 37. Smith KJ, Blakemore WF, McDonald WI: Central remyelination restores secure conduction. Nature 1979, 280:395e396
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38. Blight AR, Young W: Central axons in injured cat spinal cord recover electrophysiological function following remyelination by Schwann cells. J Neurol Sci 1989, 91:15e34 39. Sherman DL, Brophy PJ: Mechanisms of axon ensheathment and myelin growth. Nat Rev Neurosci 2005, 6:683e690 40. Funfschilling U, Supplie LM, Mahad D, Boretius S, Saab AS, Edgar J, Brinkmann BG, Kassmann CM, Tzvetanova ID, Mobius W, Diaz F, Meijer D, Suter U, Hamprecht B, Sereda MW, Moraes CT, Frahm J, Goebbels S, Nave KA: Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 2012, 485: 517e521
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Astrocyte Activation via Stat3 Signaling Determines the Balance of Oligodendrocyte versus Schwann Cell Remyelination Q35
Glaucia Monteiro de Castro,*y Natalia A. Deja,* Dan Ma,* Chao Zhao,* and Robin J.M. Franklin* From the Wellcome Trust-MRC Cambridge Stem Cell Institute, Department of Clinical Neurosciences,* University of Cambridge, Cambridge, United Kingdom; and the Department of Biosciences,y Sao Paulo Federal University, Santos, Brazil Accepted for publication May 7, 2015.
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Address correspondence to Robin J.M. Franklin or Chao Zhao, Wellcome Trust-MRC Cambridge Stem Cell Institute and Department of Clinical Neurosciences, Clifford Allbutt Building, Cambridge Biomedical Campus, University of Cambridge, Cambridge, United Kingdom. E-mail: rjf1000@ cam.ac.uk or [email protected]. uk.
Remyelination within the central nervous system (CNS) most often is the result of oligodendrocyte progenitor cells differentiating into myelin-forming oligodendrocytes. In some cases, however, Schwann cells, the peripheral nervous system myelinating glia, are found remyelinating demyelinated regions of the CNS. The reason for this peripheral type of remyelination in the CNS and what governs it is unknown. Here, we used a conditional astrocytic phosphorylated signal transducer and activator of transcription 3 knockout mouse model to investigate the effect of abrogating astrocyte activation on remyelination after lysolecithin-induced demyelination of spinal cord white matter. We show that oligodendrocyte-mediated remyelination decreases and Schwann cell remyelination increases in lesioned knockout mice in comparison with lesioned controls. Our study shows that astrocyte activation plays a crucial role in the balance between Schwann cell and oligodendrocyte remyelination in the CNS, and provides further insight into remyelination of CNS axons by Schwann cells. (Am J Pathol 2015, -: 1e10; http://dx.doi.org/ 10.1016/j.ajpath.2015.05.011)
The adult mammalian central nervous system (CNS) is remarkably efficient at replacing myelin-forming cells after primary demyelination.1 This regenerative process is called remyelination. Although in most circumstances the new myelin-forming cells are oligodendrocytes, the myelinating cells of the CNS, it also has been well established in both experimental models and clinical disease that remyelination can be mediated by Schwann cells, the myelinating cells of the peripheral nervous system.2e8 New remyelinating oligodendrocytes are generated from a population of neural progenitor cells that are distributed widely throughout the adult CNS.9e11 These cells can be identified using a range of markers, of which two commonly used markers are Pdgfra and NG2, and generally are referred to as oligodendrocyte progenitor cells (OPCs).12 In the past, it was assumed that all Schwann cells engaged in remyelinating CNS axons were derived from peripheral nervous system Schwann cells, and entered the CNS through breaches in the astrocytic glia limitans.13,14 Although this is certainly a source of some CNS Schwann cells,10 transplantation15 and
genetic fate mapping studies10 have shown that large numbers of CNS Schwann cells are derived from OPCs. What remains unclear is how and why Schwann cell remyelination occurs within the CNS. Clues are provided by the anatomic features associated with areas of the CNS in which either oligodendrocyte or Schwann cell remyelination occurs. The most consistently observed feature is the astrocyte status: oligodendrocyte remyelination occurs in regions where astrocytes are present, restoring a complete CNS glial environment, whereas Schwann cell remyelination occurs where astrocytes are absent, resulting in patches of tissue that resemble the peripheral nervous system.16e19 Indeed, the extent of Schwann cell remyelination is directly proportional to the extent of astrocyte absence. This is seen most clearly when comparing the remyelination of ethidium bromideeinduced Supported by grants from the UK Multiple Sclerosis Society, CNPq 200993/2010-0 (G.M.d.C.) and 201797/2011-9, and Ciência Sem Fronteiras. Disclosures: None declared.
Copyright ª 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2015.05.011
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spinal cord demyelination, in which there initially is extensive astrocyte loss and hence a high proportion of Schwann cell remyelination, with remyelination of lysolecithin-induced spinal cord demyelination, which is more sparing of astrocytes and hence has a smaller Schwann cell contribution to remyelination.20 The extent of Schwann cell remyelination has been manipulated experimentally using cell transplantation approaches, in which, for example, the extent of Schwann cell remyelination of ethidium bromideeinduced spinal cord demyelination can be reduced by astrocyte transplantation.21,22 However, whether astrocyte responses play a physiological role in determining the balance of central versus peripheral types of remyelination has not been tested in experiments in which the endogenous astrocyte response is altered. In this study we took advantage of the central role of phosphorylation of the transcription factor signal transducer and activator of transcription 3 (Stat3) in the astrocyte response to CNS injury23 to address the hypothesis that the proportion of Schwann cell remyelination after experimental demyelination is dependent on astrocyte activation within the lesion. By using a conditional Cre-loxP approach we were able to specifically prevent Stat3 phosphorylation in astrocytes after focal toxin-induced demyelination. We showed that not only did this lead to a reduced astrocyte response, it also led to an impairment in OPC activation and resulted in an increased level of Schwann cell remyelination and a decreased level of oligodendrocyte remyelination, thereby showing a central role of astrocyte activation in determining the nature of CNS remyelination.
Materials and Methods Animals
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The astrocyte-specific phosphorylated Stat3 (pStat3) conditional knockout mouse strain [glial fibrillary acidic protein (GFAP)-STAT3-CKO] on a C57BL6 background kindly was provided by Dr. Michael Sofroniew (University of California, Los Angeles, CA).23 Ablation of the activated form of Stat3 in astrocytes was achieved by conditional Cre-loxP recombination. In this strain, the Cre recombinase is expressed under the mouse GFAP promoter (GFAP-Cre). Recombination occurs at loxP sites flanking exon 22 (the phosphorylation site containing exon) of the Stat3 gene (Stat3fl/fl). As a result, phosphorylation of Stat3, crucial for its function, does not occur. Experimental animals were bred using homozygous Stat3fl/fl males and heterozygous Cre-expressing females (GFAP-Creþ/-:Statfl/fl). The resulting GFAP-STAT3-CKO mice showed normal development and were fertile. Both male and female mice were used in the experiments, with noneCre-expressing littermates (Statfl/fl) used as controls. Experiments were performed in compliance with UK Home Office regulations and institutional guidelines.
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Toxin-Induced Demyelination Focal spinal cord demyelination was created as previously described.24 Briefly, 8- to 10-week-old mice were anesthetized with isoflurane and the spinal cord was exposed at the level of T12/T13 by removing the soft tissue between the vertebrae. One microliter of 1% L-a-lysophosphatidylcholine (lysolecithin; Sigma-Aldrich, Gilingham, UK) in sterile saline was injected using a Hamilton syringe fitted Q9 with a fine glass tip into the ventral spinal cord white matter (Figure 1A). At designated time points after injection, mice ½F1 were terminally anesthetized with an overdose of pentobarbital before being perfused transcardially with either 4% paraformaldehyde for immunohistochemistry or 4% glutaraldehyde for resin embedding and electron microscopy.
Immunohistochemistry Immunohistochemistry was performed on 12-mmethick frozen sections. When required, heat-mediated antigen retrieval with 10 mmol/L sodium citrate buffer (pH 6) was performed before the standard protocol for indirect immunofluorescence staining. Briefly, slide-mounted sections were washed with phosphate-buffered saline (pH 7.4), and blocked with 5% normal donkey serum and 0.1% Triton X-100 in phosphate-buffered saline for 1 hour at Q10 room temperature. Sections then were incubated with primary antibodies diluted in blocking solution overnight at 4 C. The following primary antibodies were used: Olig2 1:200 (Millipore, Watford, UK), CC1 1:100 (Apc; Cal- Q11 biochem, San Diego, CA), ionized calcium binding adaptor molecule 1 (Iba1) 1:500 (Wako, Osaka, Japan), pStat3 (Tyr705) 1:100 (Cell Signaling Technology, Beverly, MA), and Aldh1l1 1:100 (clone N103/39; Neuromab, Davis, CA). For Apc and Aldh1l1 staining on lesion sections, Mouse On Mouse blocking reagents (Vector Labs) Q12 Q13 were used to reduce nonspecific background according to the manufacturer’s instructions. Staining was visualized with Alexa Fluoreconjugated secondary antibodies (1:500; Invitrogen, Paisley, UK). The slides were counterstained with 0.1% Sudan Black (Sigma-Aldrich) to reduce background and show the lesion area, and subsequently were mounted in FluorSave Reagent (Calbiochem). The images were acquired using a Zeiss Axio Observer fluorescence microscope.
In Situ Hybridization In situ hybridization of digoxigenin-labeled complementary RNA probes for myelin protein zero and myelin proteolipid protein was performed as previously described.25 Briefly, Q14 sections were hybridized with digoxigenin-labeled complementary RNA probes at 65 C overnight and subjected to a standard wash protocol (50% formamide, 1 standard saline citrate, 0.1% Tween-20, 65 C, 3 30 minutes) to remove nonspecific probe binding. The target bound probes were
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Figure 1 Expression of phosphorylated signal transducer and activator of transcription 3 (pStat3) after focal demyelination in spinal cord white matter is reduced in glial fibrillary acidic protein (GFAP) promoter-controlled conditional Stat3 knockout mice. A: Control and mutant animals were injected with lysolecithin to create a focal demyelination lesion. B and C: The quantification of the total numbers of pStat3þ (B) and pStat3/Gfap double-positive (C) cells in control and GFAP-STAT3-CKO lesions. DeI: Immunostaining of lesioned ventral spinal cord white matter at 14 days after lesion (dpl). D and G: Merged immunostaining for phosphorylated Stat3 (pStat3), and the nuclear dye Hoechst 33342. The dotted line marks the border of the demyelinated area, as demarcated by increased cellularity shown by Hoechst staining (inset in D). E and H: Co-labeling with pStat3 and astrocyte marker Gfap; boxed area in each image is magnified in panels F and I, respectively. J and K: Representative images show colocalization of an alternative astrocyte marker Aldh1l1 and pStat3 in demyelinated areas at 14 dpl, in control (J) and GFAP-STAT3-CKO (K) animals. LeO: Quantification is shown. pSTAT3 also is expressed in other types of cells, including CD11bþ macrophages/ microglia (M and N) and Olig2þ oligodendrocyte lineage cells (O) within lesions. Means SEM are shown. Arrowheads (F, J, K, and MeO) indicate cells labeled with both markers. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (DeI); 25 mm (J and K). WT, wild type.
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For each animal, three demyelinated lesion sections, separated by approximately 120 mm, were selected from within the central region of the lesion. For immunostaining, the outline of each lesion was defined based on the increase in cellularity inside the lesion, as visualized by Hoechst 33342 counterstain. For in situ hybridization, the outline was defined based on the lesioned tissue texture, using Zeiss AxioVision software. The number of Q16 marker-positive cells inside the lesions was counted manually using ImageJ version x.xxx (NIH, Bethesda, Q17 MD; http://imagej.nih.gov/ij), and normalized against the lesion area. The average of three sections was used for each lesioned animal. For each group, four to five animals were used. To compare differences between the control and experimental groups, a two-way analysis of variance followed by the Bonferroni post-test was used, and the threshold for statistical significance was set at P < 0.05.
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Electron Microscopy Animals were perfused with 4% glutaraldehyde in phosphate-buffered saline containing 0.4 mmol/L CaCl2. The spinal cord was sliced coronally at 1-mm thickness and treated with 2% osmium tetroxide overnight before being subjected to a standard protocol for epoxy resin
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Figure 3 Macrophage and microglia infiltration after demyelination in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO mice is similar to that in controls. The microglia/macrophages in the demyelinated area at 5 days after lesion (dpl) were visualized with antibody against macrophage/microglia marker ionized calcium binding adaptor molecule 1 (Iba1) in control (A) and GFAPSTAT3-CKO (B) mice. C: Mean optical density was used to compare the Iba1 immunoreactivity between controls and mutants across time points. D: The average macrophage/microglia infiltration area at different survival times. Measurements were taken from transverse sections of the lesion that had the largest lesion area (identified by increased cellularity). Means SEM. Scale bars: 50 mm (A and B).
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detected by alkaline phosphataseeconjugated antidigoxigenin antibody, and visualized as purple precipitate after incubation in NBT/BCIP solution according to the manufacturer’s instructions (Roche, Lewes, UK). The slides were dehydrated with ascending concentration of ethanol, cleared with xylene, and mounted in dibutyl phthalate in xylene. Images were acquired with the Zeiss Axio Observer microscope.
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Astrocyte response to demyelination is attenuated in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO mice. AeD: Images illustrate areas of lysolecithininduced demyelination in ventral spinal cord white matter at 14 days after lesion (dpl), immunolabeled for astrocyte marker Gfap and nucleus dye Hoechst 33342 (A and C), with lesion areas demarcated by counterstaining with Sudan Black (B and D). Graphs show the relative total area occupied by immunoreactive EeG: Gfap staining in demyelinated lesions (E) and the average length of astrocytic processes in control and GFAP-STAT3-CKO mice (F), and the number of astrocytes inside the lesion area identified by the astrocyte marker Aldh1l1 (G). Dotted lines mark the lesion boundaries. Means SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (AeD).
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The response of oligodendrocyte lineage cells to lysolecithininduced spinal cord demyelination in glial fibrillary acidic protein (GFAP)signal transducer and activator of transcription 3 (STAT3)-CKO mice. AeD: Demyelinated areas of ventral spinal cord lesions at 14 days after lesion (dpl) from control and GFAP-pSTAT3-CKO mice double-stained for oligodendrocyte lineage marker Olig2 and transcription factor Sox2, a marker for activated oligodendrocyte progenitor cells. B and D: Enlarged areas marked by an orange rectangle in panels A and C, respectively. EeG: Densities of single-labeled and co-labeled cells are compared. Arrows indicate double-labeled cells. Means SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (A and B).
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559 560 561 Expression of Phosphorylated Stat3 in Astrocytes Is 562 Reduced in GFAP-STAT3-CKO Mice after Toxin-Induced 563 CNS Demyelination 564 565 Phosphorylation of Stat3 is increased in multiple CNS cell 566 25e27 types during injury. To verify this in our demyelination 567 model we examined the expression of pStat3 in control, 568 noneCre-expressing lesioned and unlesioned mice, using a 569 pStat3-specific antibody. To obtain a focal demyelinating 570 lesion, animals were injected with 1% lysolecithin solution 571 572 into the ventral spinal cord (Figure 1A). In normal unle573 sioned spinal cord, very few cells were found to express 574 pStat3 (not shown). In contrast, there was a marked increase 575 in pStat3 expression 5 days after lesion (dpl) (Figure 1B). 576 The pStat3 levels remained increased at 14 and 21 dpl, 577 albeit they were somewhat decreased compared with the 578 expression at 5 dpl (Figure 1B). The staining was most 579 intense within the nucleus (Figure 1D). pStat3þ cells 580 included a variety of cell types, such as CD11bþ macro581 phages/microglia, Olig2þ oligodendrocyte lineage cells, and 582 Gfapþ astrocytes (Figure 1, E, F, MeO). 583 584 Stat3 phosphorylation plays a key role in mediating 585 astrocyte responses to CNS injury.23 To explore the 586 astrocyte-specific role of pStat3 in lysolecithin-induced 587 demyelination further, we used a conditional Stat3 588 knockout mouse model in which Cre recombinase was 589 expressed under the GFAP promoter. The Cre recombinase 590 excised the floxed Stat3 exon 22 containing the phosphor591 ylation site (Tyr 705) involved in Stat3 activation,23 592 resulting in a mutant unable to phosphorylate Stat3 in as593 trocytes (GFAP-STAT3-CKO mice). We first investigated 594 pStat3 expression in lesioned GFAP-STAT3-CKO mice. As 595 596 expected, the number pStat3-expressing astrocytes, as 597 examined by co-labeling with astrocyte markers Gfap 28 598 (Figure 1, CeI) and aldehyde dehydrogenase 1 (Aldh1l1) þ 599 surrounding pStat3 nuclei, was significantly lower in the 600 mutants compared with controls (Figure 1, JeL). 601 602 Demyelination-associated Astrogliosis Is Reduced in 603 604 GFAP-STAT3-CKO Mice 605 606 Lysolecithin demyelination is characterized by an abundance of astrocytes within the lesioned area (Figures 1, CeL, and 2, ½F2 607 608 AeD). In control mice, we observed an increase in Gfap 609 immunoreactivity within and beyond the demyelinated area 610 (the latter was determined by the lipophilic dye Sudan Black) 611 (Figure 2, A and B). An increase in Gfap expression also was 612 seen in demyelinated areas of the spinal cord in GFAP613 STAT3-CKO mice when compared with unlesioned tissue 614 surrounding the lesioned areas (Figures 1H, and 2, C and D), 615 although the intensity of the staining was lower than in 616 617 control animals (not shown). Because Gfap staining localizes 618 to astrocytic processes that often form a tangled mesh in 619 injured tissue, rendering quantification of individual positive 620
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621 Control 21DPL B STAT3CKO 21DPL C CC1+ cells A 622 1200 623 1000 624 800 625 600 626 400 ** 627 * 200 628 0 629 14 DPL 21 DPL CC1Hoechst CC1Hoechst 630 631 plp+ cells Control 21DPL E STAT3CKO 21DPL F 632 D 1000 633 634 800 635 600 636 400 * ** 637 200 638 639 0 plp plp 14 DPL 21 DPL 640 641 Figure 5 Oligodendrocyte remyelination is reduced in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO 642 mice. Remyelination by oligodendrocytes in control and GFAP-pSTAT3-CKO mice at 14 and 21 days after lesion (dpl) was assessed by immunostaining for the 643 differentiated oligodendrocyte marker CC1 (A and B) and in situ hybridization for proteolipid protein (plp) mRNA (D and E). Images show representative Q29 644 examples of demyelinated lesions at 21 dpl. C and F: The cell densities for each marker at selected survival times are compared. Means SEM are shown. Q30 *P < 0.01 and **P < 0.001. Scale bars: 100 mm (A, B, D, and E). 645 646 cells challenging, we instead compared the areas occupied by the conditional astrocytic pStat3 ablation on oligodendro647 reactive astrocytes in control and GFAP-STAT3-CKO spinal cyte remyelination by comparing the distribution of oligo648 649 cord lesions, as defined by a clear boundary of increased Gfap dendrocyte lineage cells using different markers expressed 650 reactivity. We found the relative area of reactive astrocytes at specific stages of lineage progression in control and 651 Q18 over total lesion area to be reduced in GFAP-STAT3-CKO mutant lesioned mice. In control animals, Olig2þ cell 652 mice at both 5 and 14 dpl (Figure 2E). This was verified by numbers increased at 5 dpl (when OPCs are recruited 653 counting the number of Aldh1l1þ cells in the demyelinated actively to the lesion), increased further at 14 dpl (when 654 differentiation is ongoing), and remained high at 21 dpl areas, which showed a significant 40% to 60% reduction in 655 (when remyelination is near completion) (Figure 4, A, B, ½F4 GFAP-STAT3-CKO mice compared with controls at all 656 Q19 three survival time points examined (Figure 2G and E). In GFAP-STAT3-CKO animals, the density of ). 657 þ Olig2 cells was comparable with controls at 5 dpl, but was 658 Reducing Stat3 Activation in Astrocytes Does Not Alter reduced significantly at 14 and 21 dpl (Figure 4, CeE). 659 Macrophage Responses to Demyelination Increased expression of the transcription factor Sox2 is a 660 661 marker of OPC activation (unpublished observations). The Abrogation of Stat3 activation results in spread of inflammation 662 density of total Sox2þ cells and Sox2þ/Olig2þ co-labeled after a spinal cord crush injury.23,29 To assess the influence of 663 cells in the demyelinated area were significantly lower in 664 astrocytic Stat3 knockout in our demyelination model, we the GFAP-STAT3-CKO group compared with controls at 5 665 examined the microglia/macrophage response in GFAPand 14 dpl, suggesting impaired OPC activation (Figure 4, F 666 STAT3-CKO mice after demyelination by immunostaining for and G). There was also a reduction in Sox2þ cells that did 667 Iba1. Iba1þ cells were present at high density throughout the not express Olig2, which are likely to be astrocytes, and this 668 ½F3 lesion (Figure 3). It was not feasible to quantify the microglia/ is consistent with the data in Figure 2G. The reduced density 669 macrophage cellular density because of the fused pattern of of Olig2þ and Olig2þ/Sox2þ cells was mirrored by a 670 immunostaining in lesions, therefore we measured the normalreduced expression of the mature oligodendrocyte markers 671 ized mean optical density to represent the extent of microglia/ CC1 and myelin proteolipid protein at 14 and 21 dpl 672 macrophage infiltration in the demyelinated area. There was no 673 (Figure 5). ½F5 difference in either Iba1 intensity or the area of Iba1þ cell 674 infiltration at all of the time points examined (Figure 3, C and D). 675 Attenuated Astrogliosis Increases Schwann Cell CNS 676 The Attenuated Astrocyte Response Reduces 677 Remyelination 678 Oligodendrocyte Remyelination 679 The mutual exclusivity of the presence of astrocytes and Q20 680 Schwann cell remyelination within the same region of repaired Astrocytes are known to influence OPCs during CNS 681 demyelinated lesions led us to reason that an attenuated remyelination.21,30e32 We therefore assessed the impact of 682
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Figure 6 Schwann cell remyelination increases in glial fibrillary acidic protein (GFAP)-signal transducer and activator of transcription 3 (STAT3)-CKO mice. Remyelinating Schwann cells from control and GFAP-STAT3-CKO mice at 21 days after lesion (dpl) were examined by periaxin immunostaining (AeD) and myelin protein zero (mpz) mRNA in situ hybridization (FeH). Dotted lines (AeD, F, and G) mark demyelinated areas. E and F: Quantification of the relative area of positive periaxin immunofluorescence (E) and cells containing mpz mRNA (F). J and K: Semithin resin sections from control (J) and STAT3CKO (K) lesioned mice were stained with toluidine blue. Areas of oligodendrocyte remyelination are marked with the yellow letter O, and that of Schwann cells are marked with the red letter S. M and N: Enlarged boxed areas from panels J and K, respectively. L and O: Examples of axons remyelinated by oligodendrocytes are marked by yellow arrowheads; green arrowheads point to examples of axons that were not demyelinated, blue arrowheads indicate poorly remyelinated axons, and red arrowheads mark typical morphology of myelinating Schwann cells. These observations were verified further by electron microscopy with examples shown in control (L) and GFAP-STAT3-CKO (O) cells. Inset (O) shows an enlarged view of the boxed area, showing the typical structure of myelinating Schwann cells. Means SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (AeD, F, G, JeM); 5 mm (N and O).
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Monteiro de Castro et al 869 astrocytic response could lead to increased Schwann cell 870 remyelination in GFAP-STAT3-CKO mutants. Indeed, 871 compared with lesioned controls, the GFAP-STAT3-CKO 872 spinal cord white matter lesions showed increased areas of 873 periaxin antigenicity and increased density of myelin protein 874 zero mRNA expression at 14 and 21 dpl, indicating increased 875 876 ½F6 Schwann cell remyelination (Figure 6, AeI). The increased number of remyelinating Schwann cells was confirmed by 877 analyzing semithin resin sections and performing electron 878 879 microscopy, where Schwann cells could be recognized readily 880 by their typical signet-ring morphology and relatively thicker 881 myelin sheath (Figure 6, JeO). Notably, in the GFAP-STAT3882 CKO lesions, Schwann cell remyelinated areas contained more 883 demyelinated axons than oligodendrocyte remyelinated areas at 884 all of the time points examined (Figure 6, M and N). Because 885 toxin-induced demyelination invariably undergoes complete 886 remyelination, it is likely that these few remaining demyelinated 887 axons eventually will undergo remyelination. 888 889 890 891 Discussion 892 893 In this study, we used a transgenic conditional astrocytic 894 pStat3 knockout model to show that altering astrocyte 895 activation significantly influences the response to CNS 896 demyelination injury. This model had been used previously 897 to study astrogliosis and scar formation in spinal cord 898 trauma.23 In accordance with this previous study, we found 899 that abrogating astrocyte activation decreased the astrocytic 900 response in the lesion. However, unlike in the trauma 901 model, we found no effect of pStat3 manipulation in as902 903 trocytes on macrophage responses.29 Crucially, our study 904 focused on remyelination and showed that oligodendrocyte 905 remyelination was reduced and Schwann cell remyelination 906 was increased in lesioned GFAP-STAT3-CKO mice. Our 907 findings therefore constitute the first direct proof of the 908 indispensable role of astrocyte activation in the balance 909 between Schwann cell and oligodendrocyte remyelination in 910 the CNS. 911 The intriguing phenomenon of Schwann cell remyelina912 tion within the CNS has been recognized for many decades, 913 however, its mechanisms and functions remain obscure. It 914 915 now is known, as a result of genetic fate mapping strategies, 916 that contrary to the previously held belief, many of the 917 Schwann cells that appear in the CNS are not immigrants 918 from the peripheral nervous system but instead are derived 919 from CNS progenitor cells.10 It also has been recognized 920 that Schwann cell remyelination often occurs around blood 921 vessels and within areas of damaged CNS from which 922 astrocytes are absent.16,19,33 This observation has led to the 923 hypothesis that the presence or absence of astrocytes de924 termines if CNS progenitor cells support remyelination of 925 demyelinated axons by becoming an oligodendrocyte or a 926 927 Schwann cell. Our results clearly show a central role for 928 astrocytes in determining the balance of central versus 929 peripheral-type remyelination. It remains unclear how 930
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astrocytes exert this effect. One proposed hypothesis is that members of the bone morphogenetic protein family induce OPCs to become Schwann cells, however, this is prevented in the presence of astrocytes by astrocyte-derived inhibitors of bone morphogenetic protein signaling, with OPCs becoming oligodendrocytes instead. However, although there is some evidence that the fate of transplanted OPCs can be influenced by prior treatment in vitro with bone morphogenetic proteins,34,35 there is no compelling evidence for such mechanisms in vivo. Because the current STAT3 knockout takes place at the first appearance of GFAP expression during development, it is possible that there are long-term changes in the environment that contribute to the shift in remyelination type. However, if Q21 such changes do exist they would seem to be shown only after injury because phenotypic changes have not been identified in the absence of injury in either our study or in previous studies on astrocyte STAT3-null animals. Thus, Q22 exactly how OPCs become Schwann cells in the CNS in the absence of astrocytes remains to be fully explored. What is the functional significance of Schwann cells myelinating CNS axons? There are two main functions of myelin: to allow rapid saltatory conduction and to help maintain axon health and integrity.36 It has been evident for many years that Schwann cell myelination restores saltatory conduction to demyelinated CNS axons, and from this perspective it appears to make no difference which type of myelin surrounds the axons.37,38 However, the relative effect of peripheral versus central-type myelin on axonal integrity is entirely unknown. Schwann cells and oligodendrocytes differ in a number of ways: they develop from different tissues, use different strategies to myelinate target axons, produce different extracellular components, and assemble molecularly distinct nodes and paranodes.39 Moreover, key differences have been shown in their metabolic relationships with the axons they ensheath.40 Therefore, it is possible that in the context of recovery from CNS demyelinating injury, Schwann cell CNS remyelination may have distinctive physiological advantages compared with oligodendrocyte remyelination, although this hypothesis remains to be tested. Certainly, in the context of immune-mediated damage directed against epitopes specific to oligodendrocytes and their myelin, one might imagine that the Schwann cell would be resistant to direct injury and therefore this form of remyelination might protect against subsequent oligodendrocyte-directed immune attack. Our study has shown that a substantial reduction of pStat3-mediated astrocyte activation is a sufficient prerequisite to sway the remyelination process toward peripheral nervous systemetype remyelination in the CNS. This evidence will help to elucidate further the development and function of peripheral-type CNS remyelination, which presents not only an intriguing biological phenomenon but also an interesting and unexplored therapeutic possibility.
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Acknowledgments We thank Dr. Michael Sofroniew for providing the GFAPSTAT3-CKO mice, and Michael Peacock and Daniel Morrison for their help with electron microscopy.
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References 1. Franklin RJM, Ffrench-Constant C: Remyelination in the CNS: from biology to therapy. Nat Rev Neurosci 2008, 9:839e855 2. Dusart I, Isacson O, Nothias F, Gumpel M, Peshanski M: Presence of Schwann cells in neurodegenerative lesions of the central nervous system. Neurosci Lett 1989, 105:246e250 3. Felts PA, Woolston AM, Fernando HB, Asquith S, Gregson NA, Mizzi OJ, Smith KJ: Inflammation and primary demyelination induced by the intraspinal injection of lipopolysaccharide. Brain 2005, 128: 1649e1666 4. Itoyama Y, Webster HD, Richardson EP Jr, Trapp BD: Schwann cell remyelination of demyelinated axons in spinal cord multiple sclerosis lesions. Ann Neurol 1983, 14:339e346 5. Snyder DH, Valsamis MP, Stone SH, Raine CS: Progressive demyelination and reparative phenomena in chronic experimental allergic encephalomyelitis. J Neuropathol Exp Neurol 1975, 34: 209e221 6. Duncan ID, Hoffman RL: Schwann cell invasion of the central nervous system of the myelin mutants. J Anat 1997, 190:35e49 7. Guest JD, Hiester ED, Bunge RP: Demyelination and Schwann cell responses adjacent to injury epicenter cavities following chronic human spinal cord injury. Exp Neurol 2005, 192:384e393 8. Blakemore WF: Remyelination by Schwann cells of axons demyelinated by intraspinal injection of 6-aminonicotinamide in the rat. J Neurocytol 1975, 4:745e757 9. Gensert JM, Goldman JE: Endogenous progenitors remyelinate demyelinated axons in the adult CNS. Neuron 1997, 19: 197e203 10. Zawadzka M, Rivers LE, Fancy SPJ, Zhao C, Tripathi R, Jamen F, Young K, Goncharevich A, Pohl H, Rizzi M, Rowitch DH, Kessaris N, Suter U, Richardson WD, Franklin RJM: CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination. Cell Stem Cell 2010, 6:578e590 11. Tripathi RB, Rivers LE, Young KM, Jamen F, Richardson WD: NG2 glia generate new oligodendrocytes but few astrocytes in a murine experimental autoimmune encephalomyelitis model of demyelinating disease. J Neurosci 2010, 30:16383e16390 12. Richardson WD, Young KM, Tripathi RB, McKenzie I: NG2-glia as multipotent neural stem cells: fact or fantasy? Neuron 2011, 70: 661e673 13. Franklin RJM, Blakemore WF: Requirements for Schwann cell migration within CNS environments: a viewpoint. Int J Dev Neurosci 1993, 11:641e649 14. Franklin RJM, Blakemore WF: Reconstruction of the glia limitans by sub-arachnoid transplantation of astrocyte-enriched cultures. Microsc Res Tech 1995, 32:295e301 15. Keirstead HS, Ben-Hur T, Rogister B, O’Leary MT, Dubois-Dalcq M, Blakemore WF: Polysialylated neural cell adhesion molecule-positive CNS precursors generate both oligodendrocytes and Schwann cells to remyelinate the CNS after transplantation. J Neurosci 1999, 19: 7529e7536 16. Blakemore WF: Invasion of Schwann cells into the spinal cord of the rat following local injections of lysolecithin. Neuropathol Appl Neurobiol 1976, 2:21e39 17. Woodruff RH, Franklin RJM: Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following
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