Death receptor 6 negatively regulates oligodendrocyte ... - Nature

Articles

Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination

© 2011 Nature America, Inc. All rights reserved.

Sha Mi1, Xinhua Lee1, Yinghui Hu1, Benxiu Ji1, Zhaohui Shao1, Weixing Yang1, Guanrong Huang1, Lee Walus1, Kenneth Rhodes1, Bang Jian Gong1, Robert H Miller2 & R Blake Pepinsky1 Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithininduced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis. Myelin provides discontinuous insulation along axons that leads to a reduction in the threshold for neuronal activation and an increase in axonal conduction velocity1. In the CNS, myelin is generated by oligodendrocytes. Oligodendrocyte development is characterized by three major stages: A2B5+ progenitor cells, immature platelet-derived growth factor receptor α (PDGFRα)+ and O4+ oligodendrocytes, and mature myelinating oligodendrocytes that express myelinassociated glycoprotein (MAG), MBP and myelin oligodendrocyte glycoprotein (MOG)1–3. The maturation of oligodendrocyte lineage cells is highly regulated during development and tightly linked to survival signals2,3. Factors that regulate oligodendrocyte progenitor cell (OPC) survival and oligodendrocyte maturation are poorly understood1,4–6. Several factors have been implicated in regulating oligodendrocyte survival, differentiation and myelination. Leucine-rich repeat and Ig domain–containing-1 (LINGO-1) is a negative regulator of OPC differentiation and axon myelination1,7–11, and blocking LINGO-1 function leads to robust myelination in lysolecithin and EAE models8,9. Tumor necrosis factor-α (TNF-α) induces oligodendrocyte death through casp3 activation in rats12, leading to myelin loss. Other growth and differentiation factors such as insulin-like growth factor 1, neuregulin, nerve growth factor, leukemia inhibitory factor and Notch/Jagged have been investigated, but their mechanisms of action remain unclear13–21. DR6 belongs to the TNF receptor (TNFR) superfamily22,23. Like other TNFR family members, DR6 contains four highly conserved cysteine-rich extracellular domains. Its cytoplasmic tail contains an ~60-residue death domain. Death domains can form homotrimeric complexes upon ligand binding that negatively regulate cell survival24.

Overexpression of DR6 in HeLa cervical carcinoma cells leads to casp3 activation and cell death in a death domain–dependent manner23. DR6-null (Tnfrsf21−/−) mice are viable, fertile and show no obvious abnormal tissue pathology25. DR6 expression in neurons leads to cell death and axon degeneration through a mechanism involving binding to N-terminal β amyloid precursor protein (N-APP)26. Here we show that DR6 is expressed in the oligodendrocyte lineage, where it acts as a negative regulator of oligodendrocyte maturation. Attenuation of DR6 function in oligodendrocytes leads to lower casp3 activation and greater survival, maturation and myelination in vitro and in two animal models of demyelination. This discovery may provide a new therapeutic avenue for the treatment of dysmyelination disorders and demyelinating diseases such as multiple sclerosis. RESULTS DR6 is expressed in the oligodendrocyte lineage To determine whether DR6 expression is developmentally regulated, we used quantitative real-time RT-PCR to quantify DR6 mRNA levels in embryonic day 18 (E18), postnatal day 1 (P1), P7, P18, P21 and adult rat brains. DR6 mRNA expression was low at E18, peaked at P7–P14 and then declined in adulthood (Fig. 1a). DR6 protein expression followed a similar time course, as determined by western blotting (Fig. 1b). We determined the expression pattern of DR6 during oligodendrocyte development by three different approaches (Fig. 1c–f). First, we detected DR6 mRNA in O4 + premyelinating oligodendrocytes in the corpus callosum of adult rat brain by in situ hybridization with a DR6 antisense probe. No signal was detected using a DR6 sense probe (Fig. 1c). Second, we assessed DR6 protein

1Biogen

Idec, Cambridge, Massachusetts, USA. 2Center for Translational Neuroscience, Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. Correspondence should be addressed to S.M. ([email protected]). Received 27 December 2010; accepted 6 April 2011; published online 3 July 2011; doi:10.1038/nm.2373

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expression with immunocytochemistry in purified rat A2B5 +, PDGFRα+ and MBP+ oligodendrocyte lineage cells with antibody to DR6 and found strong expression in immature PDGFRα+ oligo dendrocytes, moderate expression in A2B5 + OPCs and low expression in mature MBP+ oligodendrocytes (Fig. 1d). Preadsorption of the DR6-specific antibody with soluble DR6 abolished the labeling (data not shown). Third, through western blotting of DR6 protein expression in purified rat A2B5+, PDGFRα+ and MBP+ cells, we found fourfold higher expression in PDGFRα+ oligodendrocytes and twofold higher expression in A2B5+ cells than in MBP+ oligo dendrocytes (Fig. 1e,f). DR6 mediates OPC death through the casp3 pathway We used three approaches to define the biological function of DR6 in OPC differentiation, survival and maturation processes. First, we treated purified A2B5+ OPCs with DR6 siRNAs to decrease endogenous DR6, leading to a 50% reduction in DR6, five- and threefold increases in expression of the oligodendrocyte maturation markers MBP and MOG, respectively, and 85% decrease in activated casp3 (Fig. 2a). Second, we overexpressed full-length DR6 (DR6 FL) in A2B5+ OPCs by lentivirus infection to mimic ligand-independent receptor activation, leading to a twofold increase in activated casp3 protein expression (Fig. 2b). Third, we expressed a dominant-negative DR6 lacking the death domain (DR6 DN) in OPCs by lentivirus infection, leading to an eightfold increase in MBP expression and 50% lower casp3 protein expression, as compared with cells expressing DR6 FL (Fig. 2b). GFP protein expression confirmed DR6 FL and DR6 DN expression (the lentivirus coexpressed GFP protein, Fig. 2b). The number of activated casp3+ oligodendrocytes was twofold higher in cells infected with DR6 FL compared with control (Fig. 2c). Caspase inhibitor studies confirmed a specific role for casp3 in DR6-mediated cell death. Treatment of cultures overexpressing DR6 FL with a casp3 inhibitor (Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-FMK) decreased cell death by 60%. In contrast, treatment of parallel cultures with a casp6 inhibitor (Z-Val-Glu(OMe)-ILe-Asp(OMe)-CH2F) had no effect (Fig. 2d). These data indicate that DR6-induced oligodendrocyte death during maturation is directly linked to casp3, and not to the casp6 signaling pathway. In neurons, N-APP can bind to DR6 and induce casp3 activation (ref. 26). In contrast, we observed no activation of casp3 when oligodendrocytes were treated with N-APP

nature medicine VOLUME 17 | NUMBER 7 | JULY 2011

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Figure 1 DR6 is expressed in oligodendrocytes. (a) RT-PCR quantification of relative DR6 mRNA expression in rat brain at different development stages (mRNA level in sample E18 = 1). (b) Western blot analysis of DR6 protein expression in rat brain at different development ages. (c) In situ hybridization analysis of DR6 mRNA expression in adult rat corpus callosum sections. Red, probed with DR6 antisense mRNA; green, stained with antibody to O4; yellow, merge of red and green. The arrowheads indicate O4+DR6+ cells, and blue is DAPI staining. Scale bars, 15 µm. (d) Immunocytochemical analysis of DR6 protein expression in oligodendrocytes. Red, stained with antibody to DR6; green, stained with antibodies to A2B5, PDGFRα and MBP; yellow, merge of red and green. Scale bars, 95 µm. (e) Western blot analysis of DR6 protein expression in A2B5 +, PDGFRα+ and MBP+ oligodendrocyte cultures. β-actin expression was analyzed from the same samples as an internal control. (f) Quantification of DR6 protein expression from e. Data are shown as means ± s.e.m.

(Fig. 2e), suggesting that the pathways leading to casp3 activation in neurons and oligodendrocytes are different. Consistent with this hypothesis, an antibody to DR6 that blocks its biological activity on oligodendrocytes had no impact on the binding of DR6 to N-APP (data not shown). To confirm the role of DR6 in oligodendrocytes, we cultured OPCs from DR6-null (Tnfrsf21−/−) and wild-type (WT) mice for 5 d in differentiation medium and monitored their survival and maturation. We observed 2.5-fold more MBP+ mature oligodendrocytes (Fig. 2f,g) and 50% fewer activated casp3+ oligodendrocytes in cultures derived from DR6-null mice than from age-matched WT mice (Fig. 2h). These findings suggest that DR6 negatively regulates oligodendrocyte survival and maturation through the casp3 pathway by a mechanism independent of N-APP binding. Blocking DR6 promotes axon myelination in vitro The inhibitory role of DR6 on oligodendrocyte survival and maturation prompted us to screen for antibodies to DR6 that could be used in animal studies to assess DR6 function. Of 16 isolated monoclonal antibodies to DR6 that we generated, three (1D6, 5D10 and 2F2) promoted MBP expression in the primary OPC differentiation assay (Fig. 3a), and we selected 5D10 for further studies. In the differentiation assay, 5D10 treatment led to a fivefold increase in MBP+ oligodendrocytes (Fig. 3b,c) a 75% reduction in activated casp3+ oligodendrocytes (Fig. 3c), a 20-fold increase in MBP protein and a 50% reduction of activated casp3 protein (Fig. 3d). To test whether blocking DR6 promotes myelination by oligo dendrocytes, we transfected cocultures of rat primary A2B5+ OPCs and dorsal root ganglion (DRG) neurons with 200 nM DR6 siRNA, infected them with DR6 DN lentivirus at a multiplicity of infection (MOI) of 1 or treated them with 10 µg ml−1 monoclonal antibody to DR6 (5D10). We measured myelination by MBP immunocytochemistry and western blotting. Blocking DR6 function led to robust axonal myelination, as indicated by a greater number of MBP+ myelinated axon clusters as compared with corresponding control-treated cultures (Fig. 3e). Compared with control, the number of MBP+ myelinating clusters was greater by >50-fold in 5D10- or DR6 DN–treated cultures and by 20-fold in DR6 siRNA–treated cultures (Fig. 3f). Treatment with 5D10 led to dose-dependent increases in MBP and MOG expression (Fig. 3g), as determined by western blotting. Similarly, DR6 DN treatment produced increases in MBP and MOG

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Figure 2 DR6 antagonists promote A2B5+ OPC survival and differentiation. (a) Western blot analysis of MBP, MOG, cleaved casp3 and DR6 proteins in oligodendrocytes after treatment with DR6 or control siRNAs. β-actin expression was analyzed from the same samples as an internal control. (b) Western blot analysis of MBP, cleaved casp3 proteins in DR6 DN and DR6 FL lentivirus-infected A2B5 + OPC cultures. GFP expression was analyzed from the same samples as an internal control for lentivirus infection. (c) Quantification of percentage cleaved casp3+ oligodendrocytes after treatment with DR6 DN, DR6 FL and control virus. (d) Quantification of cleaved casp3+ cells in DR6 FL and control oligodendrocytes after treatment with casp3 and casp6 inhibitors. (e) Western blot analysis of cleaved casp3, MBP and MAG proteins in oligodendrocytes after treatment with N-APP or buffer control. (f) P2 oligodendrocytes from DR6 WT and DR6-null mice cultures stained with antibody to MBP. Scale bars, 25 µm. (g) Quantification of MBP+ mature oligodendrocytes in f. (h) Quantification of cleaved casp3+ oligodendrocytes from DR6 WT and null mice in P2 oligodendrocyte cultures. P values in c and d were determined by one-way analysis of variance (ANOVA followed by Tukey’s test), and in g and h using the unpaired t test. β-actin expression was analyzed from the same samples as an internal control for all western blots. Data are shown as means ± s.e.m.

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Figure 3 Inhibition of DR6 promotes oligodendrocyte survival, maturation and myelination. (a) Western blot analysis of MBP expression after P2 OPC treatment with DR6 antibodies (10 µg ml−1). (b) Immonocytochemical analysis to visualize MBP+ and cleaved casp3+ apoptotic oligodendrocytes after 5D10 or control isotype antibody treatment. Arrowheads indicate Casp3+ cells. Scale bars, 100 µm. (c) Quantification of MBP+ oligodendrocytes and cleaved casp3+ oligodendrocytes from b. (d) Western blot analysis of cleaved casp3 and MBP proteins in 5D10- and control IgG–treated OPC cultures. (e) Immunocytochemical visualization of myelination in 5D10-, DR6 DN- and DR6 siRNA-treated DRG-OPC cocultures and the corresponding control-treated cocultures. Scale bars, 50 µm. Red, MBP staining. (f) Quantitative analysis of immunocytochemical staining of MBP+ myelinated axon clusters from e. (g) Western blot analysis of MBP and MOG in cocultures treated with 5D10, DR6 FL and DR6 DN. (h) Western blot analysis of MBP, MAG and DR6 protein from WT and DR6-null mice cocultures. β-actin expression was analyzed from the same samples in g and h as an internal control. (i) Quantification of MBP+ axons from WT and DR6-null cocultures. P values in c, f and i were determined using the unpaired t test. Data are shown as means ± s.e.m.

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The EAE studies demonstrate that blocking DR6 through 5D10 treatment or genetic deletion of DR6 promotes remyelination. In EAE studies, 5D10-treated rats showed a 75% decrease in the infiltration of CD4+ T cells into the spinal cord (Supplementary Fig. 3). To determine whether the role of 5D10 in remyelination and functional recovery reflects its action on oligodendrocytes and not on the immune system, we first assessed remyelination in rat brain slice ex vivo cultures lacking an immune component. Labeling of P17 rat forebrain slices with black gold revealed extensive myelination in the corpus callosum (Fig. 5a). Lysophosphatidylcholine (LPC) treatment of the brain slices led to a rapid and persistent demyelination of corpus callosum, as visualized by the loss of black gold staining when compared with untreated (–LPC) slices (Fig. 5a). Addition of 5D10 to the LPC-treated slices led to a significant increase in black gold staining, indicating remyelination (Fig. 5a). Using Meso Scale Discovery (MSD) analysis, we found eightfold greater MAG protein in samples treated with 5D10 than in those treated with control antibody (Fig. 5b). Neither IgG isotype control monoclonal antibody nor the control treatment with antibody to PLP led to remyelination (Fig. 5a,b). Next, LPC was injected into the dorsal column of Sprague-Dawley rats, which induced a reproducible, nonimmune-mediated focal demyelination lesion. Treatment with 5D10 was started 3 d after LPC injection when lesion formation was maximal, and remyelination was assayed 7 d

antibody to PLP used as a control had no effect on OPC differentiation (Supplementary Figs. 1 and 2). In addition, 5D10 had no effect on oligodendrocyte differentiation in OPCs from DR6-null mice (Supplementary Fig. 2). These studies show that inhibition of DR6 function on oligodendrocytes promotes myelination in vitro. Inhibiting DR6 function promotes remyelination in vivo We used MOG EAE to determine whether blocking DR6 promotes remyelination and functional recovery in the setting of demyelination in vivo. We intraperitoneally injected rats with 5D10 when they reached an EAE disease score ≥0.75 (day ~15). Rats treated with 5D10 had significantly lower EAE scores than those treated with control antibody on days 23–32 after MOG immunization (Fig. 4a). We confirmed functional recovery by transcranial stimulation and measurement of nerve conduction velocity by electrophysiology (Fig. 4b). Conduction velocity was 11% greater in 5D10-treated rats than in the control treatment group. When we detected demyelination in spinal cord lumbar regions, rats treated with 5D10 had 2.5-fold greater remyelinated axons in the lesion centers versus control-treated EAE rats (Fig. 4c,d). To confirm the results from the 5D10 EAE study, we also carried out an EAE study in DR6-null mice. Using electron microscopy analysis, we found fivefold greater remyelination for DR6-null mice versus WT EAE mice (Fig. 4c,e).

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Figure 4 Antibody to DR6 promotes functional recovery and remyelination in the rat EAE model. (a) EAE clinical score measurement after 5D10 and control antibody treatment. Arrows indicate treatment regimen. (b) Descending nerve conduction velocity measurements after 5D10 and control antibody treatment. (c) Electron microscopy analysis of WT Tnfrsf21–/– remyelination in EAE rat spinal cords after 5D10 and control antibody treatments and in WT and DR6-null EAE mouse spinal cords. Remyelination is denoted by blue asterisks in lower-magnification images and red asterisks in higher-magnification images. (d,e) Quantification of myelinated axons from c. P values were determined using the unpaired t test. Data are shown as means ± s.e.m.

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Figure 5 A DR6 antagonist promotes remyelination in brain slice cultures and in the LPC-induced 150 spinal cord demyelination model. (a) Black gold staining (red) of myelinated axons in LPC-treated 100 corpus callosum brain slice cultures. Brain slices were demyelinated by LPC followed by 5D10 and control IgG treatments for 3 d. Remyelination was visualized by light microscopy. Scale bar, 200 µm. 50 (b) Quantification of myelin protein MAG by MSD analysis. P values were determined using one-way 0 analysis of variance (nonparametric tests). (c) Toluidine blue staining and electron microscopy (EM) –/– Ctrl IgG 5D10 WT Tnfrsf21 analysis of remyelination in LPC-demyelinated rat spinal cords after 5D10 and control antibody treatments and in LPC-demyelinated WT and DR6-null mouse spinal cords. Arrowheads, remyelinated axons; asterisks, axon pathology. (d,e) Quantification of myelinated axons from c. P values in d and e were determined using the unpaired t test. Data are shown as means ± s.e.m.


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Figure 6 Oligodendrocyte maturation and myelination in DR6-null mice. (a) Quantification of immature PDGFRα+ oligodendrocytes from DR6 knockout and WT corpus callosum at age P15 and P30. (b) Quantification of CC1+ mature oligodendrocytes from DR6-null and WT corpus callosum at age P15 and P30. (c) Western blot analysis of MAG and MBP protein expression in DR6-null and WT mouse brain at P15 and P30. DR6 expression was used to confirm the DR6-null mice; β-actin expression was analyzed from the same samples as an internal control. (d) Quantification of MAG and MBP protein expression in P15 mice from c. (e,f) EM showing visual (e) and quantitative (f) analysis of myelinated axon fibers of DR6 knockout and WT corpus callosum from P15 mice. Scale bars, 500 nM. (g) G ratio of myelinated axons from e. (h) Colocalization of DR6 mRNA (in situ hybridization) and Olig2+ OPCs by immunohistochemical analysis in human normal and multiple sclerosis (MS) brains. Red, probed with DR6 antisense mRNA; green, stained with antibody to Olig2; yellow, merge of red and green. Scale bars, 50 µm. (i) Quantification of DR6+Olig2+ cells in h. P values in a, b, d, f, g and i were determined using the unpaired t test. Data are shown as means ± s.e.m.

after therapy. Remyelination was determined using toluidine blue staining on 1 µm sections and by electron microscopy analysis (Fig. 5c). Toluidine blue–stained sections from 5D10-treated rats showed abundant remyelinated axons distributed throughout the lesion compared with the control antibody-treated group (Fig. 5c). Ultrastructural analy ses by electron microscopy from the centers of demyelinated lesions showed extensive remyelination (Fig. 5c) with about threefold more myelinated axons in 5D10-treated rats (Fig. 5d). We also carried out LPC studies in DR6-null mice. Robust remyelination was seen in the DR6-null mice, as determined in toluidine blue–stained 1 µm sections and by electron microscopy (Fig. 5c,e), with an eightfold increase in remyelination in DR6-null compared to WT LPC-treated mice (Fig. 5e). The LPC studies demonstrate that blocking DR6 by 5D10 or genetic deletion promotes remyelination and that remyelination in vivo is not primarily mediated through an anti-inflammatory response. Early-onset myelination in DR6-null mice We also assessed the effect of DR6 deletion on developmental oligo dendrocyte maturation and myelination in DR6-null mice and WT littermates. First, we analyzed PDGFRα+ and CC1+ cell numbers in corpus callosum. There was no difference in numbers of PDGFRα+ immature oligodendrocytes at P15, but we found a 20% reduction at P30 (Fig. 6a). Quantification of CC1+ cells showed twofold and 1.2-fold increases in CC1+ oligodendrocytes in DR6-null mice corpus callosum at P15 and P30, respectively (Fig. 6b). MBP and MAG expression determined by western blotting was sevenfold higher at P15 in DR6-null mice than in WT mice (Fig. 6c,d), and myelination determined by electron microscopy was twofold greater in the DR6-null mice (Fig. 6e,f). The myelin sheath was thicker in P15 DR6-null mice, with a 15% lower G ratio (axon diameter/myelinated fiber diameter) than in WT mice (Fig. 6e,g). At P30, there were no detectable differences in MBP

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and MAG protein expression in DR6-null mice (Fig. 6c), with a 10% increase in the proportion of myelinated axons and a similar G ratio when compared with WT mice. At 12 weeks of age, both DR6-null and WT mice had similar G ratios and similar numbers of myelinated axons (data not shown). These results are consistent with our in vitro and in vivo evidence that blocking DR6 promotes oligodendrocyte survival and precocious myelination during development. We also investigated DR6 mRNA and protein expression in multiple sclerosis versus normal brain tissue. We found fivefold more Olig2+DR6+ cells in situ in multiple sclerosis frontal lobe white matter (n = 3) compared with normal human white matter (Fig. 6h,i). Immunohistochemical analysis showed that the number of DR6+PDGFRα+ cells in multiple sclerosis lesions was twofold greater than in normal brain tissue (Supplementary Fig. 4a,b). DR6+CC1+ cells were not observed in multiple sclerosis lesions (Supplementary Fig. 4c–e). The total number of DR6+ cells was fourfold higher in multiple sclerosis white matter compared with normal human white matter, as determined by immunohistochemical analysis (Supplementary Fig. 5). Similarly, we observed more DR6+ cells and fewer DR6+CCI+ oligodendrocytes using immunohistochemistry in EAE versus normal rat brain (Supplementary Fig. 6). These data are consistent with the finding that DR6 expression is minimal in mature oligodendrocytes (Fig. 1d–f). DISCUSSION We have defined a crucial role for DR6 in oligodendrocyte biology. DR6 is expressed in all oligodendrocyte lineage stages, with elevated expression in immature PDGFRα+ oligodendrocytes. Expression of DR6 in PDGFRα+ cells is noteworthy, because immature PDGFRα+ oligodendrocytes are highly susceptible to cell death during develop ment and injury1,2. DR6 expression is developmentally regulated and



articles upregulated in diseases such as multiple sclerosis. DR6-null mice show earlier onset of brain myelination compared with WT littermates. Similar to other death receptors24, DR6-induced oligodendrocyte death requires the death domain and is linked to the casp3 signaling pathway. DR6 blockage by siRNA, antagonism by 5D10 monoclonal antibody or genetic loss of DR6 led to increased oligodendrocyte survival, maturation and myelination. Together, these studies provide strong evidence that DR6 acts as a general oligodendrocyte death regulator. Blocking DR6 with 5D10 promoted remyelination in in vivo LPCdemyelination and MOG-induced EAE models and functional recovery with increased nerve conduction velocity in the EAE model. In the EAE study, 5D10-treated rats had reduced CD4+ T cell infiltration into the spinal cord tissues, which may also have contributed to the lower EAE disease scores. However, the effects of 5D10 on remyelination in the LPC-induced demyelination rat model indicate that DR6 has a direct inhibitory role on myelination that is not mediated through an anti-inflammatory response. Distinct death receptor signaling pathways may mediate specific neuropathology depending on the target cell type27–37. DR6 induces neuronal cell death and axon degeneration by activating casp3 and casp6 pathways26. In axonal degeneration, N-APP has been implicated as a key activator of DR6 in the development of neurodegenerative pathologies such as Alzheimer’s disease26. Our studies demonstrate that DR6 has a pivotal role in the regulation of oligodendrocyte survival, maturation and myelination through a mechanism independent of N-APP. It is unclear whether other cell populations such as those in the hematopoietic lineage, whose biology is regulated by DR6, use distinct regulatory mechanisms to modulate DR6 signaling. Multiple sclerosis is an inflammation-induced neural degenerative disease and contains both autoimmune and demyelination components. Current therapies mainly target immunomodulation, but recent studies indicate that functional benefits can be achieved by promoting remyelination. For example, treatment with a LINGO-1 antagonist promotes CNS remyelination by modulating neural cells without affecting immune cells11,38. DR6 antagonists are unique in that they have the potential not only to target the autoimmune component but also to promote remyelination. The dual role of a DR6 antagonist in promoting remyelination and inhibiting autoimmune activation may represent a new approach for the treatment of CNS diseases caused by demyelination. Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/. Note: Supplementary information is available on the Nature Medicine website. AUTHOR CONTRIBUTIONS S.M. supervised all experiments and wrote the paper. X.L., Y.H., B.J., Z.S., W.Y., G.H., L.W. and B.J.G. performed experiments. K.R. and R.B.P. provided helpful discussions, and R.H.M. and R.B.P. revised the paper. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Miller, R.H. & Mi, S. Dissecting demyelination. Nat. Neurosci. 10, 1351–1354 (2007). 2. Miller, R.H. Regulation of oligodendrocyte development in the vertebrate CNS. Prog. Neurobiol. 67, 451–467 (2002).


3. Miller, R.H. et al. Patterning of spinal cord oligodendrocyte development by dorsally derived BMP4. J. Neurosci. Res. 76, 9–19 (2004). 4. Franklin, R.J. Why does remyelination fail in multiple sclerosis? Nat. Rev. Neurosci. 3, 705–714 (2002). 5. Franklin, R.J. Remyelination of the demyelinated CNS: the case for and against transplantation of central, peripheral and olfactory glia. Brain Res. Bull. 57, 827–832 (2002). 6. Franklin, R.J. & Hinks, G.L. Understanding CNS remyelination: clues from developmental and regeneration biology. J. Neurosci. Res. 58, 207–213 (1999). 7. Mi, S., Sandrock, A. & Miller, R.H. LINGO-1 and its role in CNS repair. Int. J. Biochem. Cell Biol. 40, 1971–1978 (2008). 8. Mi, S. et al. Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells. Ann. Neurol. 65, 304–315 (2009). 9. Mi, S. et al. LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis. Nat. Med. 13, 1228–1233 (2007). 10. Lee, X. et al. NGF regulates the expression of axonal LINGO-1 to inhibit oligodendrocyte differentiation and myelination. J. Neurosci. 27, 220–225 (2007). 11. Mi, S. et al. LINGO-1 negatively regulates myelination by oligodendrocytes. Nat. Neurosci. 8, 745–751 (2005). 12. Pang, Y., Cai, Z. & Rhodes, P.G. Effect of tumor necrosis factor-alpha on developing optic nerve oligodendrocytes in culture. J. Neurosci. Res. 80, 226–234 (2005). 13. Wang, S. et al. Notch receptor activation inhibits oligodendrocyte differentiation. Neuron 21, 63–75 (1998). 14. Mason, J.L. et al. Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/ remyelination. J. Neurosci. Res. 61, 251–262 (2000). 15. Guan, J. et al. Insulin-like growth factor-1 reduces postischemic white matter injury in fetal sheep. J. Cereb. Blood Flow Metab. 21, 493–502 (2001). 16. D’Ercole, A.J., Ye, P. & O’Kusky, J.R. Mutant mouse models of insulin-like growth factor actions in the central nervous system. Neuropeptides 36, 209–220 (2002). 17. Mason, J.L., Xuan, S., Dragatsis, I., Efstratiadis, A. & Goldman, J.E. Insulin-like growth factor (IGF) signaling through type 1 IGF receptor plays an important role in remyelination. J. Neurosci. 23, 7710–7718 (2003). 18. Chan, J.R. et al. NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes. Neuron 43, 183–191 (2004). 19. Stidworthy, M.F. et al. Notch1 and Jagged1 are expressed after CNS demyelination, but are not a major rate-determining factor during remyelination. Brain 127, 1928–1941 (2004). 20. Ishibashi, T. et al. Astrocytes promote myelination in response to electrical impulses. Neuron 49, 823–832 (2006). 21. Brinkmann, B.G. et al. Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system. Neuron 59, 581–595 (2008). 22. Bossen, C. et al. Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human. J. Biol. Chem. 281, 13964–13971 (2006). 23. Pan, G. et al. Identification and functional characterization of DR6, a novel death domain-containing TNF receptor. FEBS Lett. 431, 351–356 (1998). 24. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S. & Peter, M.E. Apoptosis signaling by death receptors. Eur. J. Biochem. 254, 439–459 (1998). 25. Zhao, H. et al. Impaired c-Jun amino terminal kinase activity and T cell differentiation in death receptor 6-deficient mice. J. Exp. Med. 194, 1441–1448 (2001). 26. Nikolaev, A., McLaughlin, T., O’Leary, D.D. & Tessier-Lavigne, M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457, 981–989 (2009). 27. Clarke, P., Beckham, J.D., Leser, J.S., Hoyt, C.C. & Tyler, K.L. Fas-mediated apoptotic signaling in the mouse brain following reovirus infection. J. Virol. 83, 6161–6170 (2009). 28. Swarup, V., Ghosh, J., Das, S. & Basu, A. Tumor necrosis factor receptor-associated death domain mediated neuronal death contributes to the glial activation and subsequent neuroinflammation in Japanese encephalitis. Neurochem. Int. 52, 1310–1321 (2008). 29. Reich, A., Spering, C. & Schulz, J.B. Death receptor Fas (CD95) signaling in the central nervous system: tuning neuroplasticity? Trends Neurosci. 31, 478–486 (2008). 30. Mi, S. Troy/Taj and its role in CNS axon regeneration. Cytokine Growth Factor Rev. 19, 245–251 (2008). 31. Zhou, X.F. & Li, H.Y. Roles of glial p75NTR in axonal regeneration. J. Neurosci. Res. 85, 1601–1605 (2007). 32. Yamashita, T., Fujitani, M., Yamagishi, S., Hata, K. & Mimura, F. Multiple signals regulate axon regeneration through the Nogo receptor complex. Mol. Neurobiol. 32, 105–111 (2005). 33. Shao, Z. et al. TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron 45, 353–359 (2005). 34. Park, J.B. et al. A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors. Neuron 45, 345–351 (2005). 35. Yamashita, T. & Tohyama, M. The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI. Nat. Neurosci. 6, 461–467 (2003). 36. McGee, A.W. & Strittmatter, S.M. The Nogo-66 receptor: focusing myelin inhibition of axon regeneration. Trends Neurosci. 26, 193–198 (2003). 37. Wong, S.T. et al. A p75(NTR) and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein. Nat. Neurosci. 5, 1302–1308 (2002). 38. Mi, S. et al. Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells. Ann. Neurol. 65, 304–315 (2009).

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ONLINE METHODS

In situ hybridization. Rat brain frozen sections and multiple sclerosis tissues prepared and processed as previously described39 were probed with digoxigenin-labeled DR6 antisense probe (5′-TAATACGACTCACTATAGGGGCT GGTGGGTAAGTTGTGGT-3′) and sense RNA probe (5′-ATTTAGGTGAC ACTATAGAACTCGCGGTACCTTCTCTGAC-3′).


Cell culture. We prepared purified oligodendrocyte lineage cells and DRG neurons as previously described11. We used nerve growth factor (100 ng ml−1; BD Bioscience) to inhibit basal level myelination in the coculture18. We cultured mouse P2 OPCs from WT and null mice as previously described11. To assess OPC maturation and myelination, we cultured A2B5+ OPCs in differentiation medium containing neurotrophin-3 and ciliary neurotrophic factor as previously described11. DR6 FL and DR6 DN plasmid construction and cell infection. We inserted the human DR6 FL (residues 1–655) DNA sequence into the NotI sites of HRSTIRESeGFP lentivirus vector11 using oligonucleotide primers 5′-CACGGGATC CGCGGCCGCATGGGGACCTCTCCGAGCAGC-3′ and 5′– CAGGGATCCG CGGCCGCCTACAGCAGGTCAGGAAGATGGC-3′. We inserted the human DR6 DN (residues 1–370) DNA sequence into the NotI sites of HRST-IRESeGFP lentivirus vector using oligonucleotide primers 5′-CACGGGATCCGCGGCCG CATGGGGACCTCTCCGAGCAGC-3′ and 5′-CAGGGATCCGCGGCCGCC TAGATACTGCACACCACAATCACCAC-3′. We transfected DR6 FL, DR6 DN and GFP control plasmids into 293 cells to produce lentivirus as previously described40. We infected oligodendrocytes with lentivirus at a MOI of 1. After 2 d, we washed the cultures with culture medium and mixed with DRG neurons for cocultures. The lentivirus coexpressed GFP protein, which we used to confirm oligodendrocyte infection. DR6 RNAi transfection. We obtained rat DR6 siRNAs from Dharmacon (N-056379-00) as a mixture of four siRNAs: AGAAACGGCUCCUUUA UUA, GGAAGGACAUCUAUCAGUU, GGCCGAUGAUUGAGAGAUU and GCAGUUGGAAACAGACAAA. Control scrambled siRNA had the sequence GGUGACAUGAUCGACAGCCAU. We carried out transfections using rat oligodendrocyte nucleofector kit (Amaxa) according to the manufacturer’s instructions. Generation of DR6-deficient mice. We generated C57BL/6 genetic background DR6-deficient mice at Taconic Artemis. Briefly, we replaced exons 2 and 3 of the Tnfrsf21 locus by a positive selection cassette flanked by a Flp recombination site . We generated the targeting vector using bacterial artificial chromosome (BAC) clones from the C67BL/6J RPCI-23 BAC library and transfected into C57BL/6N Taconic embryonic stem cell line. We isolated homologous recombinant clones and used them to generate chimeric animals by injection into C57BL/6 blastocyts. Replacing exons 2 and 3 with the positive selection cassette led to loss of function of the gene encoding DR6 by preventing transcription of exons 2–6. We interbred heterozygous mice to generate DR6deficient mice. We determined the genotype of the DR6 locus by PCR using tail DNA with the following primers: mutant, 5′-GGAAGTTGGTGAAACTC TGACC-3′; WT, 5′-CAGCGCCATAGTGGAAAAGG-3′; and common primers, 5′-GGTGATCCTATCTGAATTGCACC-3′. Real-time RT-PCR. We extracted mRNAs from brain tissues or cells using an RNA miniprep kit according to the manufacturer’s instructions (Stratagene). We generated cDNA from purified mRNAs (High-Capacity cDNA Archive Kit, Applied Biosystems) and used them as templates for RT-PCR analysis with primers ordered from Applied Biosystems.

nature medicine

Generation of N-APP protein. We inserted His-tagged (C-terminal) human APP (residues 1–286) into the NotI site of the pV90 vector and transfected it into HEK293 cells. We collected the culture medium and N-APP was purified with a Ni-NTA column (Qiagen). Western blots. We carried out western blotting as previously described11 using mouse antibody to MBP (SMI 94 and SMI99, 1:4,000, Covance), mouse antibody to MOG (1:500, Biogenidec), rabbit antibody to cleaved casp3 (1:1,000, Cell Signaling no. 9661), mouse antibody to DR6 (Biogenidec) and rabbit antibody to β-actin (1:2,000, Sigma no. A5060). Band intensities were quantified by densitometry. Immunohistochemistry. We fixed tissue sections or cells in 4% (wt/vol) paraformaldehyde and processed them as previously described11. We used rabbit antibody to DR6 (Santa Cruz no. SC-25772), rabbit antibody to cleaved casp3 (Cell Signaling no. 9661), mouse antibody to MBP, mouse antibody to A2B5 (Chemicom no. mAB312R), mouse antibody to Olig2 (Millipore no. mABH50) and rabbit antibody to PDGFR (Santa Cruz no. SC-338) for immunohisto chemistry. We obtained multiple sclerosis tissues from the postmortem brain frontal lobe white matter of three individuals with multiple sclerosis. We purchased paired multiple sclerosis and normal tissue from BioChain and Folio Array of Discovery. We carried out histology and electron microscopy for determining remyelination as described8,9,11. LPC-induced demyelination in rat corpus callosum slices. We carried out the brain slice culture method as described8. To measure MAG expression, brain slices were lysed in 250 µl of 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 (vol/vol) at 4 °C for 2 h. We subjected clarified lysates (25 mg) to MSD analysis using methods and reagents from Meso Scale Diagnostics. We obtained MAG capture and detection antibodies from R&D (no. DY538). Demyelination models. We carried out the LPC and EAE models as described8,9,41. All animal protocols were in accordance with US National Institutes of Health guidelines and approved by the Biogen Idec Institutional Committee. Generating antibodies to DR6. We immunized female 8-week-old RBF mice (Jackson Labs) intraperitoneally with 50 µg of soluble DR6 in complete Freund’s adjuvant (Sigma) for primary immunization and incomplete Freund’s adjuvant for boosts. We immunized mice four times at 2–3 week intervals. Mice were killed, and splenic B lymphocytes were aseptically harvested, washed and used in polyethylene glycol–mediated lymphocyte somatic cell fusions to the murine SP2/0-Ag14 myeloma. We cultured HAT-resistant hybridomas in 10% FBS (vol/vol) in DMEM and screened by ELISA and FACS for immunoreactivity specific to DR6 protein. Statistical analyses. GraphPad Prism software was used for statistical analysis. In all studies, comparison of mean values was conducted with unpaired Student’s t tests or one-way ANOVA with Tukey correction. In all analyses, statistical significance was determined at the 5% level (P < 0.05).

39. Mi, S. et al. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403, 785–789 (2000). 40. Rubinson, D.A. et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406 (2003). 41. Mi, S. et al. Rodent EAE model for the study of axon integrity and remyelination. Nat. Protoc. doi:10.1038/nprot.2007.389 (2007).

doi:10.1038/nm.2373