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• W1998475020 abstract "•ET-1 is highly upregulated in astrocytes in human MS lesions•Astrocyte-derived ET-1 inhibits remyelination•ET-1 is an endogenous regulator of Notch signaling•ET-R antagonist PD142,893 can be used therapeutically to promote remyelination Oligodendrocyte progenitor cells (OPCs) can repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. However, the OPC potential to differentiate can be prevented by inhibitory signals present in the pathological lesion environment. Identification of these signals is essential to promote OPC differentiation and lesion repair. We identified an endogenous inhibitor of remyelination, Endothelin-1 (ET-1), which is highly expressed in reactive astrocytes of demyelinated lesions. Using both gain- and loss-of-function approaches, we demonstrate that ET-1 drastically reduces the rate of remyelination. We also discovered that ET-1 acts mechanistically by promoting Notch activation in OPCs during remyelination through induction of Jagged1 expression in reactive astrocytes. Pharmacological inhibition of ET signaling prevented Notch activation in demyelinated lesions and accelerated remyelination. These findings reveal that ET-1 is a negative regulator of OPC differentiation and remyelination and is potentially a therapeutic target to promote lesion repair in demyelinated tissue. Oligodendrocyte progenitor cells (OPCs) can repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. However, the OPC potential to differentiate can be prevented by inhibitory signals present in the pathological lesion environment. Identification of these signals is essential to promote OPC differentiation and lesion repair. We identified an endogenous inhibitor of remyelination, Endothelin-1 (ET-1), which is highly expressed in reactive astrocytes of demyelinated lesions. Using both gain- and loss-of-function approaches, we demonstrate that ET-1 drastically reduces the rate of remyelination. We also discovered that ET-1 acts mechanistically by promoting Notch activation in OPCs during remyelination through induction of Jagged1 expression in reactive astrocytes. Pharmacological inhibition of ET signaling prevented Notch activation in demyelinated lesions and accelerated remyelination. These findings reveal that ET-1 is a negative regulator of OPC differentiation and remyelination and is potentially a therapeutic target to promote lesion repair in demyelinated tissue. Current multiple sclerosis (MS) therapies can be effective in patients with relapsing and remitting MS but have little impact in promoting remyelination in tissue, leading to permanently demyelinated lesions with substantial axonal loss (Buck and Hemmer, 2011Buck D. Hemmer B. Treatment of multiple sclerosis: current concepts and future perspectives.J. Neurol. 2011; 258: 1747-1762Crossref PubMed Scopus (43) Google Scholar, Compston and Coles, 2008Compston A. Coles A. Multiple sclerosis.Lancet. 2008; 372: 1502-1517Abstract Full Text Full Text PDF PubMed Scopus (3604) Google Scholar). Repair of demyelinated MS plaques is carried out by endogenous oligodendrocyte (OL) progenitor cells (OPCs) in a process called remyelination (Ffrench-Constant and Raff, 1986Ffrench-Constant C. Raff M.C. Proliferating bipotential glial progenitor cells in adult rat optic nerve.Nature. 1986; 319: 499-502Crossref PubMed Scopus (303) Google Scholar). However, several studies have shown that OPCs often fail to differentiate in chronic MS lesions (Chang et al., 2002Chang A. Tourtellotte W.W. Rudick R. Trapp B.D. Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis.N. Engl. J. Med. 2002; 346: 165-173Crossref PubMed Scopus (783) Google Scholar, Wolswijk, 1998Wolswijk G. Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells.J. Neurosci. 1998; 18: 601-609Crossref PubMed Google Scholar). The molecular mechanisms that prevent OPC maturation and OL regeneration under pathological conditions are largely unknown. OPCs migrate to demyelinated lesions, proliferate, and eventually differentiate into mature OLs to produce myelin (Franklin and Ffrench-Constant, 2008Franklin R.J. Ffrench-Constant C. Remyelination in the CNS: from biology to therapy.Nat. Rev. Neurosci. 2008; 9: 839-855Crossref PubMed Scopus (1100) Google Scholar). This transition from a progenitor cell to a myelinating OL can be negatively regulated by signals that are present in the pathological lesion environment. This is created, in part, by a dense network of reactive astrocytes (RAs) (Compston and Coles, 2008Compston A. Coles A. Multiple sclerosis.Lancet. 2008; 372: 1502-1517Abstract Full Text Full Text PDF PubMed Scopus (3604) Google Scholar, McKhann, 1982McKhann G.M. Multiple sclerosis.Annu. Rev. Neurosci. 1982; 5: 219-239Crossref PubMed Scopus (51) Google Scholar). It is still poorly understood how RAs impact OPC development and whether signals released or expressed by astrocytes limit remyelination (Moore et al., 2011Moore C.S. Abdullah S.L. Brown A. Arulpragasam A. Crocker S.J. How factors secreted from astrocytes impact myelin repair.J. Neurosci. Res. 2011; 89: 13-21Crossref PubMed Scopus (118) Google Scholar, Nair et al., 2008Nair A. Frederick T.J. Miller S.D. Astrocytes in multiple sclerosis: a product of their environment.Cell. Mol. Life Sci. 2008; 65: 2702-2720Crossref PubMed Scopus (255) Google Scholar). It is interesting that recent studies have identified the Notch activator Jagged1 as a signal expressed by RAs in MS tissue that might limit OPC differentiation and remyelination (John et al., 2002John G.R. Shankar S.L. Shafit-Zagardo B. Massimi A. Lee S.C. Raine C.S. Brosnan C.F. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation.Nat. Med. 2002; 8: 1115-1121Crossref PubMed Scopus (404) Google Scholar, Stidworthy et al., 2004Stidworthy M.F. Genoud S. Li W.W. Leone D.P. Mantei N. Suter U. Franklin R.J. Notch1 and Jagged1 are expressed after CNS demyelination, but are not a major rate-determining factor during remyelination.Brain. 2004; 127: 1928-1941Crossref PubMed Scopus (144) Google Scholar, Zhang et al., 2009Zhang Y. Argaw A.T. Gurfein B.T. Zameer A. Snyder B.J. Ge C. Lu Q.R. Rowitch D.H. Raine C.S. Brosnan C.F. John G.R. Notch1 signaling plays a role in regulating precursor differentiation during CNS remyelination.Proc. Natl. Acad. Sci. USA. 2009; 106: 19162-19167Crossref PubMed Scopus (162) Google Scholar). However, it is still unknown how Jagged1 expression or Notch activation is regulated in demyelinated lesions and whether these pathways are beneficial or detrimental to the overall remyelination process. In a previous study, we identified endothelin-1 (ET-1) as a signaling molecule synthesized in the corpus callosum (CC) following demyelinating injury (Gadea et al., 2008Gadea A. Schinelli S. Gallo V. Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway.J. Neurosci. 2008; 28: 2394-2408Crossref PubMed Scopus (183) Google Scholar). ET-1 is a secreted signaling peptide, which has systemic roles as a vasomodulator in the cardiovascular system (Rubanyi and Botelho, 1991Rubanyi G.M. Botelho L.H. Endothelins.FASEB J. 1991; 5: 2713-2720Crossref PubMed Scopus (244) Google Scholar). It is interesting that RAs produce ET-1 following various brain injuries, and we found that this peptide promotes reactive astrogliosis in demyelinated tissue (Gadea et al., 2008Gadea A. Schinelli S. Gallo V. Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway.J. Neurosci. 2008; 28: 2394-2408Crossref PubMed Scopus (183) Google Scholar, Jiang et al., 1993Jiang M.H. Höög A. Ma K.C. Nie X.J. Olsson Y. Zhang W.W. Endothelin-1-like immunoreactivity is expressed in human reactive astrocytes.Neuroreport. 1993; 4: 935-937Crossref PubMed Scopus (69) Google Scholar). Despite the abundance of ET-1 following injury, and its essential role in inducing reactive astrogliosis, the role or mechanistic action of ET-1 during remyelination has not been defined. Here, we use the well-established lysolecithin (LPC) model of focal demyelination to recapitulate some aspects of the focal lesions that are found in MS tissue. Specifically, this model allows us to investigate the time course and cell specificity of ET-1 signaling and how it regulates remyelination efficiency in vivo. Using both genetic and pharmacological approaches, we demonstrate the mechanistic action of ET-1 during remyelination. We show that astrocyte-derived ET-1 inhibits OPC differentiation and remyelination through activation of Notch signaling and that this effect can be reversed by a clinically used ET receptor (ET-R) panantagonist. Our results present a therapeutic candidate to promote repair in demyelinated lesions where OPC differentiation is stalled or limited. We have previously demonstrated that the neuropeptide ET-1 is upregulated in the CC following LPC-induced focal demyelination and that overall ET-1 levels peak at 5 days postlesion (dpl) (Gadea et al., 2008Gadea A. Schinelli S. Gallo V. Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway.J. Neurosci. 2008; 28: 2394-2408Crossref PubMed Scopus (183) Google Scholar). While we found ET-1 coexpression in glial fibrillary acidic protein positive (GFAP+) cells in the subventricular zone (SVZ) during development (Gadea et al., 2009Gadea A. Aguirre A. Haydar T.F. Gallo V. Endothelin-1 regulates oligodendrocyte development.J. Neurosci. 2009; 29: 10047-10062Crossref PubMed Scopus (63) Google Scholar), expression of ET-1 in astrocytes in LPC lesions had not been analyzed. Of the three endothelin isoforms, only ET-1 mRNA was found in the microdissected tissue from the CC and cingulum, in either saline- or LPC-injected tissue (Figures 1A and 1B ). Further ET-1 expression analysis revealed that ET-1 was specifically upregulated in GFAP+ astrocytes within LPC lesions (Figures 1D and 1E). The total number of ET-1+GFAP+ cells peaked between 3 and 7 dpl, and gradually decreased until 30 dpl, when only very few double-labeled cells were found (Figure 1C). Costaining with CD31, an endothelial cell marker, showed a small increase in the number of CD31+ET-1+ cells at 3 and 7 dpl, but there was no difference between vehicle- and LPC-injected hemispheres (Figures S1A, S1B, and S1F available online). CD31+ET-1+ cells made up approximately 22% and 14% of the total number of ET-1+ cells at 3 and 7 dpl, respectively. (Figure S1E). We found a slight upregulation of ET-1 in MAC1+ microglia (Figures S1C, S1D, and S1G), but these cells only comprised 13% and 5% of the total number of ET-1+ cells at 3 and 7 dpl, respectively. Staining was also comparatively weak as compared to ET-1 staining in astrocytes (Figure S1D). Examination of ET-1 expression in the active borders of chronic active (CA) MS lesions (Figure 1F) revealed that ET-1 was specifically upregulated in demyelination regions of the tissue (Figures 1F and 1G). In these areas, large numbers of ET-1-expressing astrocytes were found (Figure 1H). Furthermore, consistent with our previous observation that a high density of immature Olig1+ OPCs (Arnett et al., 2004Arnett H.A. Fancy S.P. Alberta J.A. Zhao C. Plant S.R. Kaing S. Raine C.S. Rowitch D.H. Franklin R.J. Stiles C.D. bHLH transcription factor Olig1 is required to repair demyelinated lesions in the CNS.Science. 2004; 306: 2111-2115Crossref PubMed Scopus (354) Google Scholar) populate the active borders of CA MS lesions (Moll et al., 2013Moll N.M. Hong E. Fauveau M. Naruse M. Kerninon C. Tepavcevic V. Klopstein A. Seilhean D. Chew L.J. Gallo V. Oumesmar B.N. SOX17 is expressed in regenerating oligodendrocytes in experimental models of demyelination and in multiple sclerosis.Glia. 2013; 61: 1659-1672Crossref PubMed Scopus (26) Google Scholar), we found large numbers of OPCs in close proximity to ET-1+ cells in the same active borders (Figure 1I). There was no evidence of ET-1 expression by immature OPCs (Figure 1I), but ET-1 expression was also found in MHCII+ T cells (data not shown). These results demonstrate that demyelination leads to an abrupt increase in ET-1 expression within the lesion. This increase in ET-1 was conserved between experimentally induced lesions in mice and in human MS tissue. The majority of ET-1 expression was found in astrocytes, demonstrating that they are the predominant source of ET-1 following injury. While the effects of ET-1 on specific cell types (including astrocytes) have been studied in detail (Schinelli, 2006Schinelli S. Pharmacology and physiopathology of the brain endothelin system: an overview.Curr. Med. Chem. 2006; 13: 627-638Crossref PubMed Scopus (69) Google Scholar), the effect of ET-1 on the important endogenous repair process following demyelination has not been examined. We have previously shown that ET-1 directly inhibits OPC differentiation in vitro and elicits promigratory effects during the development of the subcortical white matter (Gadea et al., 2009Gadea A. Aguirre A. Haydar T.F. Gallo V. Endothelin-1 regulates oligodendrocyte development.J. Neurosci. 2009; 29: 10047-10062Crossref PubMed Scopus (63) Google Scholar). Therefore, we sought to understand the potential role of ET-1 following LPC-induced demyelination in vivo using both gain- and loss-of-function approaches. First, we infused exogenous ET-1 into remyelinating lesions and measured the extent of mature OL regeneration, using the mature OL markers CC1 and MAG. We and others have found that, following LPC-induced demyelination, OPC differentiation into mature OLs begins to occur at approximately 14 dpl (Aguirre et al., 2007Aguirre A. Dupree J.L. Mangin J.M. Gallo V. A functional role for EGFR signaling in myelination and remyelination.Nat. Neurosci. 2007; 10: 990-1002Crossref PubMed Scopus (275) Google Scholar). Based on our immunohistochemical analysis (Figure 1E) and previous western blot analysis (Gadea et al., 2008Gadea A. Schinelli S. Gallo V. Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway.J. Neurosci. 2008; 28: 2394-2408Crossref PubMed Scopus (183) Google Scholar), endogenous ET-1 levels peak during the first week of remyelination and are very low at 14 dpl. Therefore, we extended the natural window of ET-1 release following LPC demyelination by infusing exogenous ET-1 beginning at 14 dpl by using miniosmotic pumps. Miniosmotic pumps containing 100 nM ET-1 were installed at 14 dpl and left until 21 dpl (Figure 2A). In the vehicle-infused LPC lesions at 21 dpl, a large number of CC1+Olig2+ and MAG+ cells were found, indicating substantial levels of repair (Figures 2B, 2D, 2E, and 2G). In contrast, in the ET-1-infused LPC lesions, a significant reduction in the number of mature OLs (CC1+Olig2+ and MAG+ cells) was found (Figures 2C, 2D, 2F, and 2G). To further characterize and label newly generated OLs, bromodeoxyuridine (BrdU) was injected once per day at 6, 7, and 8 dpl, when OPCs are proliferating within the lesion (Figure 2A). We found that, while there was little change in the number of Olig2+BrdU+ cells between the vehicle- and ET-1-infused lesions (Figure 2J), there was a significant decrease in the number of CC1+Olig2+BrdU+ cells in the ET-1-infused lesions as compared to saline controls (Figure 2K). This showed that fewer Olig2+ OPCs had matured into CC1+ OLs and that similar numbers of early OPCs were present following the infusion. Additionally, these results confirmed that the mature CC1+ cells that we observed in the vehicle-infused lesions were newly generated. Altogether, these results indicated that when the window of ET-1 release is extended into the OPC differentiation phase of remyelination (14–21 dpl), OL differentiation was delayed. RAs specifically upregulate ET-1 expression during the first week after demyelination, when OPC expansion occurs at the expense of differentiation. The vast majority of ET-1+ cells following demyelination were also RAs (Figure S1E). Therefore, we eliminated ET-1 expression in astrocytes following demyelination to specifically assess the role of astrocyte-derived ET-1 on remyelination efficiency. An ET-1flox/flox mouse was bred with an hGFAP-Cre-ERT2mouse to selectively eliminate ET-1 expression in astrocytes (hGFAP-Cre-ERT2;ET-1flox/flox mouse). First, we examined the expression of ET-1 in white matter RAs from demyelinated hGFAP-Cre-ERT2;ET-1flox/flox mice with tamoxifen or vehicle injections (Figures 3A–3C). At the peak of ET-1 expression (5 dpl), a 76% reduction was found in the total number of ET-1+GFAP+ cells in the lesion (Figures 3B and 3C). Infusion of exogenous ET-1 during remyelination limited OPC differentiation, so we measured OPC maturation following genetic ablation of ET-1 in astrocytes to determine the effects on OPC development. Three experimental groups were established: (1) ET-1 fl/fl Creneg + tamoxifen, (2) ET-1 fl/fl Cre+ + vehicle, and (3) ET-1 fl/fl Cre+ + tamoxifen. It is interesting that we found a significant increase in the number of CC1+Olig2+ (Figures 3D–3F and 3J) and MAG+ cells (Figures 3G–3I and 3K) in the ET-1 fl/fl Cre+ + tamoxifen mice, as compared to ET-1 fl/fl Creneg + tamoxifen and ET-1 fl/fl Cre+ + vehicle littermates. We also observed an increase in the CC1+/NG2+ ratio in the ET-1 fl/fl Cre+ + tamoxifen mice as compared to controls (Figure 3L). Altogether, these results demonstrate that selective deletion of ET-1 in astrocytes significantly increased the number of mature OLs in LPC lesions after 2 weeks and shifted the OL ratio from an immature to mature phenotype. Conversely, extended expression of ET-1 during remyelination led to a reduction in the number of mature OLs generated in LPC lesions. These findings indicate that astrocyte-derived ET-1 acts as an inhibitor of OPC differentiation and remyelination. We wanted to identify the mechanisms by which ET-1 acts to limit OPC maturation. There are two potential mechanisms of ET-1 action: (1) direct signaling to OPCs through ET-Rs and (2) indirect signaling to OPCs through astrocytes. We have previously shown that ET-1 can act directly on OPCs to limit their differentiation by activation of ET-Rs on their cell surface, particularly during migration (Gadea et al., 2009Gadea A. Aguirre A. Haydar T.F. Gallo V. Endothelin-1 regulates oligodendrocyte development.J. Neurosci. 2009; 29: 10047-10062Crossref PubMed Scopus (63) Google Scholar). Therefore, we wanted to investigate the effect of ET-1 signaling through astrocytes and the resulting effects of OPC differentiation. We previously identified ET-1 as a potent activator of astrocytes (Gadea et al., 2008Gadea A. Schinelli S. Gallo V. Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway.J. Neurosci. 2008; 28: 2394-2408Crossref PubMed Scopus (183) Google Scholar), but expression of signals by those RAs that inhibit OPC differentiation following ET-1 exposure was not explored. It has been previously described that RAs in MS lesions express high levels of Jagged1, a ligand for the Notch1 receptor (John et al., 2002John G.R. Shankar S.L. Shafit-Zagardo B. Massimi A. Lee S.C. Raine C.S. Brosnan C.F. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation.Nat. Med. 2002; 8: 1115-1121Crossref PubMed Scopus (404) Google Scholar). In fact, elevated Jagged1 expression by astrocytes was found in the active borders of CA MS lesions (John et al., 2002John G.R. Shankar S.L. Shafit-Zagardo B. Massimi A. Lee S.C. Raine C.S. Brosnan C.F. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation.Nat. Med. 2002; 8: 1115-1121Crossref PubMed Scopus (404) Google Scholar), the same areas where we found high ET-1 expression in astrocytes and a high density of immature OPCs (Figure 1G). In the same study, Notch1 expression was also found on OPCs. Independent studies have also shown that Notch1 inhibits OPC differentiation during both development and remyelination (Genoud et al., 2002Genoud S. Lappe-Siefke C. Goebbels S. Radtke F. Aguet M. Scherer S.S. Suter U. Nave K.A. Mantei N. Notch1 control of oligodendrocyte differentiation in the spinal cord.J. Cell Biol. 2002; 158: 709-718Crossref PubMed Scopus (171) Google Scholar, Zhang et al., 2009Zhang Y. Argaw A.T. Gurfein B.T. Zameer A. Snyder B.J. Ge C. Lu Q.R. Rowitch D.H. Raine C.S. Brosnan C.F. John G.R. Notch1 signaling plays a role in regulating precursor differentiation during CNS remyelination.Proc. Natl. Acad. Sci. USA. 2009; 106: 19162-19167Crossref PubMed Scopus (162) Google Scholar). We tested the functional relevance of Jagged1/Notch1 signaling as a possible mechanism underlying the effects of ET-1 on OPC differentiation during remyelination. First, we sought to determine whether ET-1 has a role in regulating the expression of Jagged1. In primary cultured astrocyte monolayers, ET-1 induced a significant increase in Jagged1 protein expression after 48 hr (Figure 4A). These increases in Jagged1 were blocked by preincubation with the ET-R panantagonist PD142,893 (Figures 4A and 4B). These results indicate that astrocytes express increased levels of Jagged1 following ET-1 exposure and that this increase is mediated by ET-Rs. It has been previously shown that components of Notch signaling are present in both MS lesions and in experimentally induced LPC lesions in mice (John et al., 2002John G.R. Shankar S.L. Shafit-Zagardo B. Massimi A. Lee S.C. Raine C.S. Brosnan C.F. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation.Nat. Med. 2002; 8: 1115-1121Crossref PubMed Scopus (404) Google Scholar, Stidworthy et al., 2004Stidworthy M.F. Genoud S. Li W.W. Leone D.P. Mantei N. Suter U. Franklin R.J. Notch1 and Jagged1 are expressed after CNS demyelination, but are not a major rate-determining factor during remyelination.Brain. 2004; 127: 1928-1941Crossref PubMed Scopus (144) Google Scholar). Specifically, it was shown that astrocytes express Jagged1 and OPCs express Notch1. However, to our knowledge, functional activation of Notch signaling in demyelinated lesions has not been directly demonstrated. We utilized the transgenic Notch reporter (TNR) mouse, in which enhanced green fluorescent protein (EGFP) is expressed on canonical Notch activation (Mizutani et al., 2007Mizutani K. Yoon K. Dang L. Tokunaga A. Gaiano N. Differential Notch signalling distinguishes neural stem cells from intermediate progenitors.Nature. 2007; 449: 351-355Crossref PubMed Scopus (414) Google Scholar), to study the cell specific activation and time course of Notch signaling following demyelination. As demonstrated in previous studies (John et al., 2002John G.R. Shankar S.L. Shafit-Zagardo B. Massimi A. Lee S.C. Raine C.S. Brosnan C.F. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation.Nat. Med. 2002; 8: 1115-1121Crossref PubMed Scopus (404) Google Scholar, Stidworthy et al., 2004Stidworthy M.F. Genoud S. Li W.W. Leone D.P. Mantei N. Suter U. Franklin R.J. Notch1 and Jagged1 are expressed after CNS demyelination, but are not a major rate-determining factor during remyelination.Brain. 2004; 127: 1928-1941Crossref PubMed Scopus (144) Google Scholar), Jagged1 was specifically upregulated 5-fold in astrocytes in LPC lesions at 7 dpl (Figures 4C and 4D). Further analysis of Notch component expression in microdissected white matter (WM) tissue revealed increases in Notch intracellular cleaved domain (NICD), Jagged1, and GFAP protein levels (Figures 4E–4H). Increases in NICD levels (Figures 4E and 4F) coincided with elevated Jagged1 expression at 7 dpl (Figures 4E and 4G). In the same set of experiments, we also observed high levels of GFAP expression at 7 dpl (Figures 4E and 4H), which indicated that the highest levels of astrogliosis coincide with maximal Notch activation. We have previously shown that total ET-1 expression peaks at 5 dpl (Gadea et al., 2008Gadea A. Schinelli S. Gallo V. Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway.J. Neurosci. 2008; 28: 2394-2408Crossref PubMed Scopus (183) Google Scholar), and the greatest number of ET-1-expressing astrocytes peaks between 3 and 7 dpl (Figure 1E), which, as predicted, immediately precedes the peak of Notch activation at 7 dpl. Analysis of functional Notch activation in the TNR mouse supported our earlier results. EGFP expression was specifically upregulated in LPC lesions (Figure 4I), with the greatest number of EGFP+ cells found at 7 dpl (Figure 4J). Costaining of these EGFP+ cells with Hes1, a direct downstream target of the CSL/CBF1/RPGJ transcriptional regulators, confirmed the reliability of the TNR mouse as an indicator of canonical Notch activation (Figure S2A). Colocalization analysis demonstrated that approximately half of the EGFP+ cells were Olig2+ (Figures 4K and 4M) and that, as expected, the majority of these cells were NG2+ OPCs (Figures 4K and 4L). It is interesting that we also found a significant population of EGFP+IBA1+ microglia in the core of the lesion (Figure 4K). Little or no Notch activation was found in astrocytes (GFAP) (Figure 4K). Altogether, these results demonstrate that Notch signaling is activated at high levels in demyelinated tissue and that astrocytes are likely a major regulator of Notch activation in these lesions. ET-1 also acts to promote Jagged1 expression in astrocytes, indicating that it might regulate Notch activation in vivo. Components of both the ET-1 and Notch signaling pathways are activated during the first week following demyelination, with the peak of ET-1 expression immediately preceding increases in GFAP and Jagged1 levels, indicating a possible functional connection. We showed in vitro that ET-1 strongly upregulated Jagged1 expression in astrocytes and that these increases could be blocked using PD142,893 (Figures 4A and 4B). We also found that activation of Notch signaling in vivo coincided with increases in Jagged1 expression in astrocytes in demyelinated lesions (Figures 4C, 4D, 4E, and 4G). Therefore, we hypothesized that, by blocking ET-R activation using PD142,893, we could reduce Notch activation in vivo. To test this hypothesis, PD142,893 (50 μM) was infused into the remyelinating lesions of TNR mice using miniosmotic pumps (Figures 5A and 5B ), when Notch activation remained high in OPCs (Figure 5C, bottom panels). As predicted, strong reductions were found in total Notch activation following PD142,893 infusion (Figures 5C–5E and 5F). Notch activation was recovered when recombinant Jagged1 Fc (2 μg/ml) was added to a mixture containing PD142,893 (Figure 5F). No overall changes in the total number of OL lineage cells caused by infusion of the antagonist were observed between groups (Figure 5G). To further confirm that ET-1 induces Jagged1 expression, leading to Notch activation, Jagged1 protein expression was examined in the hGFAP-Cre-ERT2;ET-1flox/flox mouse. Jagged1 levels were significantly reduced at 7 dpl in microdissected tissue from the ET-1 fl/fl Cre+ + tamoxifen mice, as compared to ET-1 fl/fl Creneg + tamoxifen and ET-1 fl/fl Cre+ + vehicle littermates (Figures 5H and 5I). These results demonstrate the functional connection of ET-1 and Notch signaling in vivo. PD142,893 strongly blocked Notch activation in LPC lesions, and exogenous Jagged1 rescues Notch activation, even when ET-1 signaling is blocked. We also confirm in our hGFAP-Cre-ERT2;ET-1flox/flox that a reduction in ET-1 produced by astrocytes is sufficient to reduce overall Jagged1 levels in the lesion. These findings also confirm that ET-1 signaling is upstream of Notch activation during remyelination. ET-1 signaling inhibits remyelination and limits OPC differentiation. We propose that one mechanism regulating this effect is expression of Jagged1 by astrocytes and resulting Notch activation in OPCs. To specifically assess the functional interaction between astrocytes and OPCs, we used a coculture system. We have shown that ET-1 promotes Jagged1 expression in astrocytes and that ET-1 signaling promotes Notch activation in vivo. To assess Notch activation in cocultures, OPCs from the TNR mouse were plated on ET-1 pretreated astrocytes. There was a significant increase in the number of EGFP+NG2+ cells in ET-1-pretreated cocultures, as compared to control cultures, indicating enhanced Notch signaling in OPCs (Figures 6A–6D). This signaling activation was blocked by preincubation with PD142,893 (Figures 6A–6D). To ensure that Notch activation was mediated by astrocyte-OPC contact and not soluble factors, cocultures were performed in which TNR mouse OPCs were plated on glass coverslips rather than in direct contact with astrocytes (Figure S3A). This prevented the cell-to-cell contact required for Notch receptor/ligand interaction but still allowed soluble factors to be released into the cell culture media. Under these conditions, very few EGFP+NG2+ OPCs were found, with no difference between the control or ET-1-pretreated groups (Figure 6D). Analysis of mature OL formation in these cocultures revealed a drastic reduction in the number of mature O1+ OLs, when plated on astrocytes pretreated with ET-1, as compared to untreated astrocytes (Figures 6E and 6F). Furthermore, a reduction in the O1+/NG2+ (mature/immature) cell ratio was seen in ET-1-pretreated cocultures, indicating a delay in OL lineage progression (Figure 6I). This effect was blocked by PD142,893 preincubation (Figures 6G and 6I). To ensure that OPC development was not directly influenced by ET-1 remaining in the culture media following astrocyte pretreatment, an anti-ET-1 antibody was added to the culture media dur" @default.
• W1998475020 created "2016-06-24" @default.
• W1998475020 creator A5005098377 @default.
• W1998475020 creator A5006763108 @default.
• W1998475020 creator A5029089948 @default.
• W1998475020 creator A5044487737 @default.
• W1998475020 creator A5048412151 @default.
• W1998475020 creator A5063495794 @default.
• W1998475020 creator A5086049459 @default.
• W1998475020 date "2014-02-01" @default.
• W1998475020 modified "2023-10-18" @default.
• W1998475020 title "Astrocyte-Derived Endothelin-1 Inhibits Remyelination through Notch Activation" @default.
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