OTUB 1 inhibits CNS autoimmunity by preventing IFN ‐γ‐induced hyperactivation of astrocytes
2019; Springer Nature; Volume: 38; Issue: 10 Linguagem: Inglês
10.15252/embj.2018100947
ISSN1460-2075
AutoresXu Wang, Floriana Mulas, Wenjing Yi, Anna Brunn, Gopala Nishanth, Sissy Just, Ari Waisman, Wolfgang Brück, Martina Deckert, Dirk Schlüter,
Tópico(s)NF-κB Signaling Pathways
ResumoArticle3 April 2019free access Source DataTransparent process OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes Xu Wang Corresponding Author [email protected] orcid.org/0000-0001-8428-9339 Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Floriana Mulas Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Wenjing Yi Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Anna Brunn Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Gopala Nishanth Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Sissy Just Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Ari Waisman orcid.org/0000-0003-4304-8234 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Wolfgang Brück Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Martina Deckert Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Dirk Schlüter Corresponding Author [email protected] orcid.org/0000-0003-1478-3328 Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Organ-specific Immune Regulation, Helmholtz-Center for Infection Research, Braunschweig, Germany Search for more papers by this author Xu Wang Corresponding Author [email protected] orcid.org/0000-0001-8428-9339 Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Floriana Mulas Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Wenjing Yi Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Anna Brunn Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Gopala Nishanth Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Sissy Just Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Ari Waisman orcid.org/0000-0003-4304-8234 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Wolfgang Brück Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Martina Deckert Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Dirk Schlüter Corresponding Author [email protected] orcid.org/0000-0003-1478-3328 Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Organ-specific Immune Regulation, Helmholtz-Center for Infection Research, Braunschweig, Germany Search for more papers by this author Author Information Xu Wang *,1,2, Floriana Mulas1,2, Wenjing Yi1,2, Anna Brunn3, Gopala Nishanth1,2, Sissy Just1,2, Ari Waisman4, Wolfgang Brück5, Martina Deckert3 and Dirk Schlüter *,1,2,6 1Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, Magdeburg, Germany 2Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany 3Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 4Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany 5Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany 6Organ-specific Immune Regulation, Helmholtz-Center for Infection Research, Braunschweig, Germany *Corresponding author. Tel: +49 391 67-13353; Fax: +49 391 67-13384; E-mail: [email protected] *Corresponding author. Tel: +49 511 532-6770; Fax: +49 511 532-4366; E-mail: [email protected] EMBO J (2019)38:e100947https://doi.org/10.15252/embj.2018100947 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Astrocytes are critical regulators of neuroinflammation in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Growing evidence indicates that ubiquitination of signaling molecules is an important cell-intrinsic mechanism governing astrocyte function during MS and EAE. Here, we identified an upregulation of the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) in astrocytes during MS and EAE. Mice with astrocyte-specific OTUB1 ablation developed more severe EAE due to increased leukocyte accumulation, proinflammatory gene transcription, and demyelination in the spinal cord as compared to control mice. OTUB1-deficient astrocytes were hyperactivated in response to IFN-γ, a fingerprint cytokine of encephalitogenic T cells, and produced more proinflammatory cytokines and chemokines than control astrocytes. Mechanistically, OTUB1 inhibited IFN-γ-induced Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling by K48 deubiquitination and stabilization of the JAK2 inhibitor suppressor of cytokine signaling 1 (SOCS1). Thus, astrocyte-specific OTUB1 is a critical inhibitor of neuroinflammation in CNS autoimmunity. Synopsis The deubiquitinating enzyme OTUB1 regulates cell signaling by blocking ubiquitin transfer from E2 conjugating enzymes to E3 ligases or by cleavage of ubiquitin chains from target proteins, but the in vivo cell type-specific function of OTUB1 is largely unknown. During CNS autoimmunity, human and murine astrocytes upregulate OTUB1, which limits CNS pathology by inhibiting IFN-γ-induced activation of the JAK2/STAT1 pathway and concomitant chemokine and cytokine production in astrocytes. OTUB1 expression of astrocytes is induced in the CNS of treatment-naïve patients with multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. OTUB1 inhibits IFN-γ-induced JAK2/STAT1 signaling by K48 deubiquitination and stabilization of the JAK2 inhibitor SOCS1. OTUB1-dependent inhibition of the JAK2/STAT1 signaling limits IFN-γ-induced cytokine and chemokine production of astrocytes. OTUB1 expression in astrocytes ameliorates the clinical severity of murine experimental autoimmune encephalomyelitis by limiting astrocytic cytokine and chemokine production, recruitment of encephalitogenic T cells to the CNS, and demyelination. Introduction Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are autoimmune diseases of the central nervous system (CNS) characterized by infiltration of inflammatory cells, demyelination, and axonal damage. Interferon (IFN)-γ-producing T helper (Th)1, interleukin (IL)-17-producing Th17, and granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing CD4+ T cells are shown to be key mediators of EAE, and all of them can induce EAE individually (Stromnes et al, 2008; Domingues et al, 2010; Codarri et al, 2011). Upon immunization with myelin oligodendrocyte glycoprotein (MOG) peptide, myelin-reactive T cells are primed in lymphatic organs and enter the subarachnoid/perivascular space through the choroid plexus or the leptomeningeal vessels (wave 1), where they are reactivated by antigen-presenting cells including dendritic cells and macrophages (Engelhardt & Sorokin, 2009; Engelhardt et al, 2016). Subsequently, T cells undergo clonal expansion and produce proinflammatory cytokines, including IFN-γ, IL-17, and tumor necrosis factor (TNF), which stimulate CNS-resident cells. Activated astrocytes produce large amounts of leukocyte-recruiting chemokines and cytokines leading to an explosive recruitment of leukocytes to the CNS (wave 2) that is associated with clinical EAE onset and demyelination (Sofroniew, 2015). In astrocytes, IL-17- and TNF-induced NF-κB activation and IFN-γ-mediated JAK-STAT1 signaling are crucial for proinflammatory gene induction and EAE development. Astrocyte-specific overexpression of the NF-κB inhibitor IκBα-dn or deletion of pivotal molecules of NF-κB and JAK-STAT1 signaling pathways including Act1, IKK2, NEMO, and IFN-γR, respectively, ameliorates EAE (van Loo et al, 2006; Brambilla et al, 2009; Kang et al, 2010; Ding et al, 2015). Of note, activation of NF-κB and JAK-STAT1 signaling is tightly controlled by ubiquitination. Ubiquitination is an important posttranslational modification in which ubiquitin, a protein consisting of 76 amino acids, is covalently attached to target proteins (Mevissen & Komander, 2017). Ubiquitination is catalyzed by a cascade of three different ubiquitinating enzymes: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s; Deshaies & Joazeiro, 2009). Ubiquitination of a substrate can be implemented by either single ubiquitin molecules (monoubiquitination or multi-monoubiquitination) or a chain of polyubiquitin molecules (polyubiquitination), which are covalently linked by one of the seven lysine residues, i.e., K6, K11, K27, K29, K33, K48, and K63. Another type of polyubiquitination, the linear polyubiquitination, is generated via linkage between the carboxyl group of an ubiquitin molecule and an N-terminal Met residue of another ubiquitin monomer (Hrdinka & Gyrd-Hansen, 2017). Functionally, ubiquitination was initially identified as a mechanism for protein degradation. However, it also participates in non-degradative cellular activities including membrane trafficking, endocytosis, and, in particular, signal transduction. K48 polyubiquitination triggers the degradation of target proteins by the 26S proteasome. In contrast, linear and K63 polyubiquitination is required for the activation of multiple signaling molecules and pathways (Hu & Sun, 2016). Ubiquitination is a reversible process and can be counter-regulated by deubiquitinating enzymes (DUBs; Mevissen & Komander, 2017). Previously, we have shown that A20, a DUB belonging to the ovarian tumor domain protease family, inhibits EAE by suppressing proinflammatory gene production in astrocytes (Wang et al, 2013a). OTU domain, ubiquitin aldehyde binding 1 (OTUB1), another DUB of the ovarian tumor domain protease family, can reduce both K48 and K63 polyubiquitination, although it has a preference for K48 polyubiquitin chains (Edelmann et al, 2009). OTUB1 regulates various signaling pathways by deubiquitinating and stabilizing multiple molecules including c-IAP1, SMAD2/3, p53, MDMX, FOXM1, and YB-1 (Sun et al, 2012; Goncharov et al, 2013; Herhaus et al, 2013; Dong et al, 2015; Wang et al, 2016; Chen et al, 2017). A recent study showed that OTUB1 increases Tau stability by removing K48-linked polyubiquitin chains from this protein in neurons (Wang et al, 2017a). However, so far, the function of OTUB1 in CNS autoimmune disease has not been studied. Here, we demonstrate that astrocytes upregulate OTUB1 protein expression in MS and EAE. To decipher the role of astrocyte-derived OTUB1 in EAE, we generated conditional GFAP-Cre OTUB1fl/fl mice that are specifically deficient of OTUB1 in astrocytes. As compared to OTUB1fl/fl mice, GFAP-Cre OTUB1fl/fl mice developed significantly more severe EAE, which was caused by increased proinflammatory gene production in OTUB1-deficient astrocytes. We further demonstrate that OTUB1 inhibited IFN-γ-induced JAK-STAT1 signaling in astrocytes by K48 deubiquitination and stabilization of SOCS1, a key inhibitor of JAK-STAT1 signaling. Thus, the present study identifies OTUB1 as a key regulator of astrocyte activation, further stressing the decisive role of astrocytes in the development of CNS autoimmunity. Results OTUB1 expression is induced in astrocytes during MS Several studies have demonstrated the regulation of important signaling pathways by OTUB1 in vitro, but the in vivo function of OTUB1 is largely unknown. To decipher a potential function of OTUB1 in vivo in CNS autoimmunity, we first analyzed OTUB1 protein expression in white matter brain biopsy tissue of treatment-naïve MS patients by immunohistochemistry. Interestingly, in all of 10 MS cases (Table EV1) analyzed, activated astrocytes adjacent to inflammatory demyelination expressed OTUB1 in the nucleus and cytoplasm (Fig 1A and B). Some oligodendrocytes also expressed OTUB1 (Fig EV1A), whereas neurons were absent from the white matter biopsy tissue. Since brain biopsy tissue from healthy individuals is not available for comparison, we studied the peritumoral area of three brain biopsies from patients with astrocytoma (WHO grade II; Table EV1) for OTUB1 expression. In contrast to neurons, resting and activated astrocytes located in the peritumoral brain tissue did not express OTUB1 (Fig EV1B). Figure 1. Upregulation of OTUB1 in astrocytes in MS lesions A, B. Activated GFAP+ astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 (arrows). Photomicrographs are obtained from case 9 (A) and case 5 (B) listed in Table EV1. Data are representative for all 10 MS patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC); original magnification ×400; scale bars correspond to 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Single oligodendrocytes express OTUB1 in MS, while astrocytes do not express OTUB1 in the peritumoral tissue of astrocytoma NogoA+ oligodendrocytes in a white matter MS lesion (case 5, Table EV1) with inflammatory infiltrates and demyelination express OTUB1 (arrows). This pattern is representative for all 10 patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488); original magnification ×400; scale bar corresponds to 50 μm. In the gray matter and the subcortical region adjacent to an astrocytoma (WHO grade II), GFAP+ astrocytes do not express OTUB1 (arrows). Neurons are OTUB1-positive (asterisks). Data are representative for three cases of peritumoral tissue of astrocytomas (WHO grade II). Double immunofluorescence with mouse anti-GFAP (FITC) and rabbit anti-OTUB1 (Cy3); original magnification ×400; scale bar corresponds to 50 μm. Download figure Download PowerPoint To study the in vivo function of OTUB1, we generated OTUB1fl/fl mice, in which exons 2 and 3 of OTUB1 were flanked by LoxP sites (Appendix Fig S1A). OTUB1fl/fl mice were crossed with Rosa26-Cre mice to delete OTUB1 in all cells and with Nestin-Cre mice to delete OTUB1 in neuroectodermal cells including astrocytes, neurons, and oligodendrocytes. Of note, mice lacking OTUB1 in all cells or only in neuroectodermal cells were embryonic lethal (Appendix Fig S1B and C). To further study the function of OTUB1 in the CNS, we selectively deleted OTUB1 in astrocytes. In these GFAP-Cre OTUB1fl/fl mice, OTUB1 expression in astrocytes was efficiently removed (Fig 2A and B). These mice were born in a normal Mendelian ratio and reached adulthood without obvious CNS defects including a normal myelination in the brain and spinal cord (Fig 2C). In addition, OTUB1 deletion in astrocytes did not result in spontaneous neuroinflammation and preserved the low numbers of leukocytes in the CNS (Figs 2D and E, and EV2A). Furthermore, astrocytic OTUB1-deficiency did not alter the composition of major leukocyte populations in the lymph node and spleen (Fig EV2B and C). Figure 2. Characterization of astrocyte-specific OTUB1 knockout mice WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Normal CNS architecture in OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Myelination is normal in the brain and spinal cord of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. CV-LFB staining; original magnification ×50; scale bars correspond to 100 μm. All photographs are representative of three mice per group. Absolute numbers of CD45+ cells in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice (n = 5 for both groups, mean + SEM). Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry (n = 5 for both groups, mean + SEM). In an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse, OTUB1 is strongly expressed by NeuN+ neurons in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC). Single NogoA+ oligodendrocytes in the brain and spinal cord (arrows) of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse express OTUB1. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488). GFAP+ astrocytes are of normal morphology and distribution in the brain and spinal cord of a non-immunized OTUB1fl/fl mouse and a GFAP-Cre OTUB1fl/fl mouse. Note that GFAP+ astrocytes of an OTUB1fl/fl mouse do not express OTUB1. GFAP+ astrocytes of a GFAP-Cre OTUB1fl/fl mouse are also OTUB1-negative and represent a negative control for OTUB1 staining. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). Data information: (F–H) Original magnification ×400; the inserts show high magnification (×1,200) of the cells marked by an arrow in the respective figures. Scale bars correspond to 50 μm, and data are representative of three mice per group. Source data are available online for this figure. Source Data for Figure 2 [embj2018100947-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Normal composition of leukocytes in GFAP-Cre OTUB1fl/fl mice A. CD45+ leukocytes were isolated from the spinal cord of GFAP-Cre OTUB1fl/fl mice and control mice by Percoll gradients. The percentages of CD4+ T cells (CD4+ CD3+), CD8+ T cells (CD8+ CD3+), B cells (CD19+ B220+), dendritic cells (CD11c+), inflammatory monocytes (Ly6Chigh CD11b+), and macrophages (F4/80+ CD11b+) were analyzed by flow cytometry. Representative dot plots are shown. B, C. CD45+ leukocytes were isolated from the lymph node (B) and spleen (C) of GFAP-Cre OTUB1fl/fl mice (n = 4) and control mice (n = 4). Cells were counted with the hemocytometer and analyzed by flow cytometry. Data show the absolute number of indicated cell populations (mean + SEM). Download figure Download PowerPoint A detailed morphological analysis of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice revealed that (i) OTUB1 was expressed by NeuN+ neurons and single Nogo2A+ oligodendrocytes in the normal brain and spinal cord of both mouse strains and (ii) neurons and oligodendrocytes were morphologically normal in GFAP-Cre OTUB1fl/fl mice (Fig 2F and G). Noteworthy, astrocytes of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were of normal morphology and distribution throughout the brain and spinal cord as assessed by their regular GFAP expression and OTUB1fl/fl astrocytes consistently lacked OTUB1 expression (Fig 2H). Ablation of OTUB1 in astrocytes aggravates EAE Since OTUB1 regulates immunologically important signaling pathways (Li et al, 2010; Goncharov et al, 2013; Herhaus et al, 2013; Peng et al, 2014) and was strongly expressed in activated astrocytes of MS lesions (Fig 1A and B), we investigated its expression and function in astrocytes after EAE induction. Upon immunization with MOG35–55 peptide and a pertussis toxin boost, OTUB1 expression was induced in activated spinal cord astrocytes of OTUB1fl/fl mice but not of GFAP-Cre OTUB1fl/fl mice at day 15 p.i. (Fig 3A). At this stage of EAE, both strains of mice harbored large inflammatory infiltrates in the spinal cord (Fig 3B). At later stages of EAE, i.e., day 22 p.i., OTUB1 expression in astrocytes strongly declined in OTUB1fl/fl mice and, as expected, remained negative in GFAP-Cre OTUB1fl/fl mice (Fig 3C). Interestingly, inflammation regressed in parallel in OTUB1fl/fl mice as indicated by the reduced leukocyte infiltration and demyelination, whereas inflammatory infiltrates and demyelination persisted in GFAP-Cre OTUB1fl/fl mice (Fig 3D). In contrast to the upregulation of OTUB1 expression in astrocytes, OTUB1 expression of NeuN+ neurons and NogoA+ oligodendrocytes remained unchanged in both OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice (Fig EV3A and B). Figure 3. Upregulation of OTUB1 in astrocytes limits EAE severity Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk). Inflammatory infiltrates in the spinal cord (encircled by a dotted line) of an OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mouse at day 15 p.i. At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk). At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control littermates (n = 29) by MOG35–55 peptide immunization with pertussis toxin. Graph represents data pooled from four experiments with seven to eight mice per group and shows the mean clinical scores ± SEM. Statistical analysis was performed using Mann–Whitney U-test; *P < 0.05. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 12) and OTUB1fl/fl control littermates (n = 12) by MOG35–55 peptide immunization without pertussis toxin. Graph represents the mean clinical scores ± SEM. Statistical analysis was performed using Mann–Whitney U-test; *P < 0.05. Data information: (A–D) All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 μm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. OTUB1 expression in the brain and spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 22 p.i NeuN+ neurons of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse equally express OTUB1 (arrows) in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC). Some NogoA+ oligodendrocytes (arrows) in the brain and spinal cord express OTUB1 in an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488). Data information: All photographs are representative of three mice per group; original magnification ×400; the inserts show higher magnification of cells marked by an arrow (×1,200). Scale bars correspond to 50 μm. Download figure Download PowerPoint In good agreement with the different kinetic of inflammation, GFAP-Cre OTUB1fl/fl mice showed significantly worsened course of EAE symptoms (Fig 3E) with an earlier disease onset (Table 1), significantly higher maximal clinical scores (Table 1), and significantly enlarged areas of demyelination in the spinal cord (Table 2) as compared to OTUB1fl/fl mice. Interestingly, numbers of GFAP+ astrocytes were also significantly increased in GFAP-Cre OTUB1fl/fl mice at days 15 and 22 p.i. (Table 3), indicating that OTUB1 expression in astrocytes critically regulates their activation. Table 1. OTUB1 deletion in astrocytes expedites disease onset and exacerbates symptoms of EAE Genotype Disease incidenceaa EAE was induced by MOG35–55 peptide immunization with pertussis toxin in GFAP-Cre OTUB1fl/fl mice (n = 29, pooled from four experiments with seven to eight mice per group) and OTUB1fl/fl control littermates (n = 29, pooled from four experiments with seven to eight mice per group). Data show absolute numbers of mice with disease versus all mice of one group and additionally the percentage of mice with disease. Day of disease onsetbb The day of disease onset was defined as the first day with a clinical score of at least 0.5. Data show the mean ± SD of the day of disease onset of GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control mice (n = 29). Maximal clinical scorecc The maximal clinical score of each mouse was recorded. Data show the mean ± SD of the maximal clinical score of GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control mice (n = 29). OTUB1fl/fl 26/29 (89.7%) 16.2 ± 1.2 2.2 ± 0.2 GFAP-Cre OTUB1fl/fl 29/29 (100%) 13.2 ± 0.8 3.0 ± 0.1 Statisticsdd Differences between GFAP-Cre OTUB1fl/fl and OTUB1fl/fl were analyzed using Mann–Whitney U-test (for maximal clinical score) and Kaplan–Meier survival curve followed by Gehan–Breslow–Wilcoxon test (for day of disease onset). P < 0.05 P < 0.05 a EAE was induced by MOG35–55 peptide immunization with pertussis toxin in GFAP-Cre OTUB1fl/fl mice (n = 29, pooled from four experiments with seven to eight mice per group) and OTUB1fl/fl control littermates (n = 29, pooled from four experiments with seven to eight mice per group). Data show absolute numbers of mice with disease versus all mice of one group and additionally the percentage of mice with disease. b The day of disease onset was defined as the first day with a clinical score of at least 0.5. Data show the mean ± SD of the day of disease onset of GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control mice (n = 29). c The maximal clinical score of each mouse was recorded. Data show the mean ± SD of the maximal clinical score of GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control mice (n = 29). d Differences between GFAP-Cre OTUB1fl/fl and OTUB1fl/fl were analyzed using Mann–Whitney U-test (for maximal clinical score) and Kaplan–Meier survival curve followed by Gehan–Breslow–Wilcoxon test (for day of disease onset). Table 2. OTUB1 reduces demyelination in the spinal cord of mice with EAE Genotype Demyelinated area (%)aa Cervical spinal cord serial cross sections (≥ 10 per mouse) were stained with CV-LFB. The demyelinated area was defined as the area lacking CV-LFB staining and was measured using ZEN imaging software. The demyelinated area is expressed as the percentage of the total spinal cord area of the same section, which is set as 100%.
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