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A new mouse model of inflammatory cortical demyelination
Abstracts
639 A new mouse model of inflammatory cortical demyelination Nielsen Lagumersindez Denis, Claudia Wrzos, Wolfgang Brück, Christine Stadelmann, Stefan Nessler Institute of Neuropathology, University Medical Centre Göttingen (UMG), Göttingen, Germany Cortical damage is prominent in multiple sclerosis (MS) and associated with disease progression and cognitive impairment. Although subpial cortical demyelination is more specific for MS than demyelination of the white matter, little is known about the pathogenic requirements for inflammatory cortical demyelination. This study combines transgenic mice on a C57BL/6 background with a stereotactic targeting approach to characterize the immunological mechanisms of inflammatory cortical demyelination. Cortical demyelination as assessed by immunohistochemistry against myelin basic protein (MBP), was most pronounced at day 5 after lesion induction and paralleled by a significant reduction of p25+ adult and Olig2 + precursor oligodendrocytes. Time course experiments demonstrated the rapid resolution of cortical demyelination and a significant decrease of perivascular intracortical demyelination was already observable at day 20 after lesion induction. The number of APP+ axons was significantly higher around perivascularly demyelinated vessels, while the density of cortical neurons was not reduced. The inflammatory cortical infiltrate was composed of inflammatory monocytes, T cells, polymorphonuclear cells (PMNs) and natural killer (NK) cells. We depleted each cell population genetically or by antibody mediated cell depletion to assess their relevance for inflammatory cortical demyelination. A pathogenic antibody response and inflammatory monocytes were required for subpial cortical demyelination. Perivascular cortical demyelination was required in addition to encephalitogenic T cells and was augmented by NK cells. Adoptive transfer studies in RAG mice demonstrated that the antigen presenting functions of B cells were dispensable. In summary, we developed and characterized a novel inflammatory cortical demyelination model. doi:10.1016/j.jneuroim.2014.08.316
636 Human N-methyl-d-aspartate receptor antibodies alter memory and behavior in a passive ventricular murine infusion model Frank Leypoldta, Jesus Planagumaa, Francesco Mannaraa, Javier Gutierrez Cuestab, Elena Martin-Garciab, Esther Aguilara, Ankit Jainc, Maarten J. Titulaerd, Rita Balice-Gordonc, Francesc Grausa, Rafael Maldonadob, Josep Dalmaua,c a IDIBAPS, Neurology, Hospital Clinic, Barcelona, Spain; bLaboratori de Neurofarmacología, Universitat Pompeu Fabra, Barcelona, Spain; c University of Pennsylvania, Department of Neuroscience, Philadelphia, United States; dDepartment of Neurology, Erasmus Medical Center, Rotterdam, Netherlands
Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a disorder that causes neuropsychiatric symptoms with prominent memory and behavioral impairment. Antibodies react with the N-terminal domain of the GluN1 subunit leading to selective and reversible internalization of cell-surface NMDAR. These effects and the frequent response to immunotherapy have suggested an antibody-
119
mediated pathogenesis, but to date there is no animal model showing that patients' antibodies cause memory and behavioral deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps, which delivered a continuous infusion of patients or control CSF (flow rate 0.25 μl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviors (sucrose preference test), depressive-like behaviors (tail suspension, and forced swimming tests), anxiety (black and white and elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical activity assessment). Here we show that infusion of patients' CSF, but not control CSF, led to progressive memory impairment, anhedonia and depressivelike behavior without affecting other behavioral or locomotor tasks. These symptoms resolved following the end of infusion. Accompanying brain studies confirmed infusion time dependent increase of brain-bound human IgG predominantly in the hippocampus. Acidextracted IgG from hippocampus of patients' CSF treated animals reacted with GluN1-expressing HEK cells. Confocal microscopy and immunoblot analysis of the effects of antibodies in the hippocampus showed decrease of clusters of total and synaptic NMDAR and NMDAR protein concentration. This downregulation inversely correlated with the intensity of hippocampal IgG deposits, did not affect the synaptic marker PSD95, and only minimally decreased cellsurface AMPAR. These molecular effects, which paralleled behavioral symptoms, gradually reversed after the end of patients' CSF infusion. Overall, the current findings establish a link between memory and behavioral deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders. doi:10.1016/j.jneuroim.2014.08.317
394 Interferon-gamma-producing natural killer cells are pathogenic in experimental autoimmune encephalomyelitis by promoting M1 macrophage activation and VLA-4 expression on CD4+ T cells Niamh Mc Guinnessa,b, Lara S. Dunganb, Marina A. Lyncha, Kingston H.g. Millsb a
Trinity College Institute of Neuroscience, University of Dublin, Trinity College Dublin, Dublin, Ireland; bImmune Regulation Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, University of Dublin, Trinity College, Dublin, Ireland Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS), where inflammation of the central nervous system (CNS) is infiltrating auto-antigen specific IL-17producing Th17 cells and interferon-gamma (IFN-gamma)-producing Th1 cells. However, cells of the innate immune system, including gamma delta T cells, natural killer (NK) cells and macrophages are also involved. Here we found that NK cells infiltrate the CNS early in disease and provide an innate source of IFN-gamma before the onset of clinical signs, whereas Th1 and Th17 cells are present at high numbers in the CNS at the peak of disease. Depletion of NK cells with anti-asialo GM1 at the induction of EAE delayed the onset of clinical signs of EAE. This correlated with a reduction in the infiltration of Th1 and Th17 cells into the CNS. The migration of Th1 and Th17 cells into the CNS and their resulting encephalitogenic activity is mediated in part by VLA-4 which
639 A new mouse model of inflammatory cortical demyelination Nielsen Lagumersindez Denis, Claudia Wrzos, Wolfgang Brück, Christine Stadelmann, Stefan Nessler Institute of Neuropathology, University Medical Centre Göttingen (UMG), Göttingen, Germany Cortical damage is prominent in multiple sclerosis (MS) and associated with disease progression and cognitive impairment. Although subpial cortical demyelination is more specific for MS than demyelination of the white matter, little is known about the pathogenic requirements for inflammatory cortical demyelination. This study combines transgenic mice on a C57BL/6 background with a stereotactic targeting approach to characterize the immunological mechanisms of inflammatory cortical demyelination. Cortical demyelination as assessed by immunohistochemistry against myelin basic protein (MBP), was most pronounced at day 5 after lesion induction and paralleled by a significant reduction of p25+ adult and Olig2 + precursor oligodendrocytes. Time course experiments demonstrated the rapid resolution of cortical demyelination and a significant decrease of perivascular intracortical demyelination was already observable at day 20 after lesion induction. The number of APP+ axons was significantly higher around perivascularly demyelinated vessels, while the density of cortical neurons was not reduced. The inflammatory cortical infiltrate was composed of inflammatory monocytes, T cells, polymorphonuclear cells (PMNs) and natural killer (NK) cells. We depleted each cell population genetically or by antibody mediated cell depletion to assess their relevance for inflammatory cortical demyelination. A pathogenic antibody response and inflammatory monocytes were required for subpial cortical demyelination. Perivascular cortical demyelination was required in addition to encephalitogenic T cells and was augmented by NK cells. Adoptive transfer studies in RAG mice demonstrated that the antigen presenting functions of B cells were dispensable. In summary, we developed and characterized a novel inflammatory cortical demyelination model. doi:10.1016/j.jneuroim.2014.08.316
636 Human N-methyl-d-aspartate receptor antibodies alter memory and behavior in a passive ventricular murine infusion model Frank Leypoldta, Jesus Planagumaa, Francesco Mannaraa, Javier Gutierrez Cuestab, Elena Martin-Garciab, Esther Aguilara, Ankit Jainc, Maarten J. Titulaerd, Rita Balice-Gordonc, Francesc Grausa, Rafael Maldonadob, Josep Dalmaua,c a IDIBAPS, Neurology, Hospital Clinic, Barcelona, Spain; bLaboratori de Neurofarmacología, Universitat Pompeu Fabra, Barcelona, Spain; c University of Pennsylvania, Department of Neuroscience, Philadelphia, United States; dDepartment of Neurology, Erasmus Medical Center, Rotterdam, Netherlands
Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a disorder that causes neuropsychiatric symptoms with prominent memory and behavioral impairment. Antibodies react with the N-terminal domain of the GluN1 subunit leading to selective and reversible internalization of cell-surface NMDAR. These effects and the frequent response to immunotherapy have suggested an antibody-
119
mediated pathogenesis, but to date there is no animal model showing that patients' antibodies cause memory and behavioral deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps, which delivered a continuous infusion of patients or control CSF (flow rate 0.25 μl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviors (sucrose preference test), depressive-like behaviors (tail suspension, and forced swimming tests), anxiety (black and white and elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical activity assessment). Here we show that infusion of patients' CSF, but not control CSF, led to progressive memory impairment, anhedonia and depressivelike behavior without affecting other behavioral or locomotor tasks. These symptoms resolved following the end of infusion. Accompanying brain studies confirmed infusion time dependent increase of brain-bound human IgG predominantly in the hippocampus. Acidextracted IgG from hippocampus of patients' CSF treated animals reacted with GluN1-expressing HEK cells. Confocal microscopy and immunoblot analysis of the effects of antibodies in the hippocampus showed decrease of clusters of total and synaptic NMDAR and NMDAR protein concentration. This downregulation inversely correlated with the intensity of hippocampal IgG deposits, did not affect the synaptic marker PSD95, and only minimally decreased cellsurface AMPAR. These molecular effects, which paralleled behavioral symptoms, gradually reversed after the end of patients' CSF infusion. Overall, the current findings establish a link between memory and behavioral deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders. doi:10.1016/j.jneuroim.2014.08.317
394 Interferon-gamma-producing natural killer cells are pathogenic in experimental autoimmune encephalomyelitis by promoting M1 macrophage activation and VLA-4 expression on CD4+ T cells Niamh Mc Guinnessa,b, Lara S. Dunganb, Marina A. Lyncha, Kingston H.g. Millsb a
Trinity College Institute of Neuroscience, University of Dublin, Trinity College Dublin, Dublin, Ireland; bImmune Regulation Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, University of Dublin, Trinity College, Dublin, Ireland Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS), where inflammation of the central nervous system (CNS) is infiltrating auto-antigen specific IL-17producing Th17 cells and interferon-gamma (IFN-gamma)-producing Th1 cells. However, cells of the innate immune system, including gamma delta T cells, natural killer (NK) cells and macrophages are also involved. Here we found that NK cells infiltrate the CNS early in disease and provide an innate source of IFN-gamma before the onset of clinical signs, whereas Th1 and Th17 cells are present at high numbers in the CNS at the peak of disease. Depletion of NK cells with anti-asialo GM1 at the induction of EAE delayed the onset of clinical signs of EAE. This correlated with a reduction in the infiltration of Th1 and Th17 cells into the CNS. The migration of Th1 and Th17 cells into the CNS and their resulting encephalitogenic activity is mediated in part by VLA-4 which