Rudolf Martini

Publications of Rudolf Martini

• Colony-stimulating factor-1 mediates macrophage-related neural damage in a model for Charcot-Marie-Tooth disease type 1X.

  Authors: Janos Groh, Joachim Weis, Hanna Zieger, E Richard Stanley, Heike Heuer, Rudolf Martini

  Brain : a journal of neurology. 11/2011; 135(Pt 1):88-104.

  Previous studies in our laboratory have shown that in models for three distinct forms of the inherited and incurable nerve disorder, Charcot-Marie-Tooth neuropathy, low-grade inflammation implicatingPrevious studies in our laboratory have shown that in models for three distinct forms of the inherited and incurable nerve disorder, Charcot-Marie-Tooth neuropathy, low-grade inflammation implicating phagocytosing macrophages mediates demyelination and perturbation of axons. In the present study, we focus on colony-stimulating factor-1, a cytokine implicated in macrophage differentiation, activation and proliferation and fostering neural damage in a model for Charcot-Marie-Tooth neuropathy 1B. By crossbreeding a model for the X-linked form of Charcot-Marie-Tooth neuropathy with osteopetrotic mice, a spontaneous null mutant for colony-stimulating factor-1, we demonstrate a robust and persistent amelioration of demyelination and axon perturbation. Furthermore, functionally important domains of the peripheral nervous system, such as juxtaparanodes and presynaptic terminals, were preserved in the absence of colony-stimulating factor-1-dependent macrophage activation. As opposed to other Schwann cell-derived cytokines, colony-stimulating factor-1 is expressed by endoneurial fibroblasts, as revealed by in situ hybridization, immunocytochemistry and detection of β-galactosidase expression driven by the colony-stimulating factor-1 promoter. By both light and electron microscopic studies, we detected extended cell-cell contacts between the colony-stimulating factor-1-expressing fibroblasts and endoneurial macrophages as a putative prerequisite for the effective and constant activation of macrophages by fibroblasts in the chronically diseased nerve. Interestingly, in human biopsies from patients with Charcot-Marie-Tooth type 1, we also found frequent cell-cell contacts between macrophages and endoneurial fibroblasts and identified the latter as main source for colony-stimulating factor-1. Therefore, our study provides strong evidence for a similarly pathogenic role of colony-stimulating factor-1 in genetically mediated demyelination in mice and Charcot-Marie-Tooth type 1 disease in humans. Thus, colony-stimulating factor-1 or its cognate receptor are promising target molecules for treating the detrimental, low-grade inflammation of several inherited neuropathies in humans.

• Attenuation of MCP-1/CCL2 expression ameliorates neuropathy in a mouse model for Charcot-Marie-Tooth 1X.

  Authors: Janos Groh, Kristina Heinl, Bianca Kohl, Carsten Wessig, Juliane Greeske, Stefan Fischer, Rudolf Martini

  Human molecular genetics. 09/2010; 19(18):3530-43.

  The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been previously shown to be an important mediator of macrophage-related neural damage in models of two distinct inheritedThe chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been previously shown to be an important mediator of macrophage-related neural damage in models of two distinct inherited neuropathies, Charcot-Marie-Tooth (CMT) 1A and 1B. In mice deficient in the gap junction protein connexin 32 (Cx32def), an established model for the X-chromosome-linked dominant form of CMT (CMT1X), we investigated the role of the chemokine in macrophage immigration and neural damage by crossbreeding the Cx32def mice with MCP-1 knockout mutants. In Cx32def mutants typically expressing increased levels of MCP-1, macrophage numbers were strongly elevated, caused by an MCP-1-mediated influx of haematogenous macrophages. Curiously, the complete genetic deletion of MCP-1 did not cause reduced macrophage numbers in the nerves due to compensatory proliferation of resident macrophages. In contrast, and as already seen in other CMT models, heterozygous deletion of MCP-1 led to reduced numbers of phagocytosing macrophages and an alleviation of demyelination. Whereas alleviated demyelination was transient, axonal damage was persistently improved and even robust axonal sprouting was detectable at 12 months. Other axon-related features were alleviated electrophysiological parameters, reduced muscle denervation and atrophy, and increased muscle strength. Similar to models for CMT1A and CMT1B, we identified MEK-ERK signalling as mediating MCP-1 expression in Cx32-deficient Schwann cells. Blocking this pathway by the inhibitor CI-1040 caused reduced MCP-1 expression, attenuation of macrophage increase and amelioration of myelin- and axon-related alterations. Thus, attenuation of MCP-1 upregulation by inhibiting ERK phosphorylation might be a promising approach to treat CMT1X and other so far untreatable inherited peripheral neuropathies in humans.

• Lack of evidence for a pathogenic role of T-lymphocytes in an animal model for Charcot-Marie-Tooth disease 1A.

  Authors: Bianca Kohl, Janos Groh, Carsten Wessig, Heinz Wiendl, Antje Kroner, Rudolf Martini

  Neurobiology of disease. 04/2010; 38(1):78-84.

  We have previously shown that in two distinct models for inherited neuropathies of the Charcot-Marie-Tooth (CMT) type, T-lymphocytes are critically involved in demyelination. In the present study, weWe have previously shown that in two distinct models for inherited neuropathies of the Charcot-Marie-Tooth (CMT) type, T-lymphocytes are critically involved in demyelination. In the present study, we tested whether T-lymphocytes have a similar pathogenetic impact in another CMT model, i.e., in mice overexpressing the peripheral myelin protein (PMP)-22, representing the most prevalent form CMT1A. By cross breeding the myelin mutant mice with mutants lacking mature T- and B-lymphocytes (RAG-1-deficient mice), the pathological alterations were not changed in comparison to PMP22 mutants with a normal immune system. Reciprocal enhancement of lymphocyte activation, by inactivation of the lymphocytic co-inhibitor programmed death-1, also did not alter pathological changes, as opposed to models with approved lymphocytic involvement. These findings strongly suggest that lymphocytes are not pathogenetically relevant in this model for CMT1A. We suggest that - in contrast to myelin phagocytosing macrophages - T-lymphocytes are not a promising target for treatment of CMT1A.

• MCP-1/CCL2 modifies axon properties in a PMP22-overexpressing mouse model for Charcot-Marie-tooth 1A neuropathy.

  Authors: Bianca Kohl, Stefan Fischer, Janos Groh, Carsten Wessig, Rudolf Martini

  The American journal of pathology. 03/2010; 176(3):1390-9.

  Charcot-Marie-Tooth 1A (CMT1A) neuropathy, the most common inherited peripheral neuropathy, is primarily caused by a gene duplication for the peripheral myelin protein-22 (PMP22). In an accordantCharcot-Marie-Tooth 1A (CMT1A) neuropathy, the most common inherited peripheral neuropathy, is primarily caused by a gene duplication for the peripheral myelin protein-22 (PMP22). In an accordant mouse model, we investigated the role of monocyte chemoattractant protein-1 (MCP-1/CCL2) as a regulator of nerve macrophages and neural damage including axonopathy and demyelination. By generating PMP22tg mice with reduced levels or lack of MCP-1/CCL2, we found that MCP-1/CCL2 is involved in the increase of macrophages in mutant nerves. PMP22tg mice with wild-type levels of MCP-1/CCL2 showed strong macrophage increase in the diseased nerves, whereas either 50% reduction or total absence of MCP-1/CCL2 led to a moderate or a strong reduction of nerve macrophages, respectively. Interestingly, MCP-1/CCL2 expression level and macrophage numbers were correlated with features indicative of axon damage, such as maldistribution of K+ channels, reduced compound muscle action potentials, and muscle weakness. Demyelinating features, however, were most highly reduced when MCP-1/CCL2 was diminished by 50%, whereas complete lack of MCP-1/CCL2 showed an intermediate demyelinating phenotype. We also identified the MEK1/2-ERK1/2-pathway as being involved in MCP-1/CCL2 expression in the Schwann cells of the CMT1A model. Our data show that, in a CMT1A model, MCP-1/CCL2 activates nerve macrophages, mediates both axon damage and demyelination, and may thus be a promising target for therapeutic approaches.

• Ectopic T-cell specificity and absence of perforin and granzyme B alleviate neural damage in oligodendrocyte mutant mice.

  Authors: Antje Kroner, Chi Wang Ip, Johannes Thalhammer, Klaus-Armin Nave, Rudolf Martini

  The American journal of pathology. 02/2010; 176(2):549-55.

  In transgenic mice overexpressing the major myelin protein of the central nervous system, proteolipid protein, CD8+ T-lymphocytes mediate the primarily genetically caused myelin and axon damage. InIn transgenic mice overexpressing the major myelin protein of the central nervous system, proteolipid protein, CD8+ T-lymphocytes mediate the primarily genetically caused myelin and axon damage. In the present study, we investigated the cellular and molecular mechanisms underlying this immune-related neural injury. At first, we investigated whether T-cell receptors (TCRs) are involved in these processes. For this purpose, we transferred bone marrow from mutants carrying TCRs with an ectopic specificity to ovalbumin into myelin mutant mice that also lacked normal intrinsic T-cells. T-lymphocytes with ovalbumin-specific TCRs entered the mutant central nervous system to a similar extent as T-lymphocytes from wild-type mice. However, as revealed by histology, electron microscopy and axon- and myelin-related immunocytochemistry, these T-cells did not cause neural damage in the myelin mutants, reflecting the need for specific antigen recognition by cytotoxic CD8+ T-cells. By chimerization with bone marrow from perforin- or granzyme B (Gzmb)-deficient mice, we demonstrated that absence of these cytotoxic molecules resulted in reduced neural damage in myelin mutant mice. Our study strongly suggests that pathogenetically relevant immune reactions in proteolipid protein-overexpressing mice are TCR-dependent and mediated by the classical components of CD8+ T-cell cytotoxicity, perforin, and Gzmb. These findings have high relevance with regard to our understanding of the pathogenesis of disorders primarily caused by genetically mediated oligodendropathy.

• A key role for gp130 expressed on peripheral sensory nerves in pathological pain.

  Authors: Manfred Andratsch, Norbert Mair, Cristina E Constantin, Nadja Scherbakov, Camilla Benetti, Serena Quarta, Christian Vogl, Claudia A Sailer, Nurcan Uceyler, Johannes Brockhaus, Rudolf Martini, Claudia Sommer, Hanns Ulrich Zeilhofer, Werner Müller, Rohini Kuner, John B Davis, Stefan Rose-John, Michaela Kress

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 10/2009; 29(43):13473-83.

  Interleukin-6 (IL-6) is a key mediator of inflammation. Inhibitors of IL-6 or of its signal transducing receptor gp130 constitute a novel class of anti-inflammatory drugs, which raise great hopes forInterleukin-6 (IL-6) is a key mediator of inflammation. Inhibitors of IL-6 or of its signal transducing receptor gp130 constitute a novel class of anti-inflammatory drugs, which raise great hopes for improved treatments of painful inflammatory diseases such as rheumatoid arthritis. IL-6 and gp130 may enhance pain not only indirectly through their proinflammatory actions but also through a direct action on nociceptors (i.e., on neurons activated by painful stimuli). We found indeed that the IL-6/gp130 ligand-receptor complex induced heat hypersensitivity both in vitro and in vivo. This process was mediated by activation of PKC-delta via Gab1/2/PI(3)K and subsequent regulation of TRPV1, a member of the transient receptor potential (TRP) family of ion channels. To assess the relevance of this direct pain promoting effect of IL-6, we generated conditional knock-out mice, which lack gp130 specifically in nociceptors, and tested them in models of inflammatory and tumor-induced pain. These mice showed significantly reduced levels of inflammatory and tumor-induced pain but no changes in immune reactions or tumor growth. Our results uncover the significance of gp130 expressed in peripheral pain sensing neurons in the pathophysiology of major clinical pain disorders and suggest their use as novel pain relieving agents in inflammatory and tumor pain.

• Accelerated Course of Experimental Autoimmune Encephalomyelitis in PD-1-Deficient Central Nervous System Myelin Mutants.

  Authors: Antje Kroner, Nicholas Schwab, Chi Wang Ip, Sonja Ortler, Kerstin Göbel, Klaus-Armin Nave, Mathias Mäurer, Rudolf Martini, Heinz Wiendl

  The American journal of pathology. 06/2009;

  It is assumed that the onset and course of autoimmune inflammatory central nervous system (CNS) disorders (eg, multiple sclerosis) are influenced by factors that afflict immune regulation as well asIt is assumed that the onset and course of autoimmune inflammatory central nervous system (CNS) disorders (eg, multiple sclerosis) are influenced by factors that afflict immune regulation as well as CNS vulnerability. We challenged this concept experimentally by investigating how genetic alterations that affect myelin (primary oligodendrocyte damage in PLPtg mice) and/or T-cell regulation (deficiency of PD-1) influence both the onset and course of an experimental autoimmune CNS inflammatory disease [MOG35-55-induced experimental autoimmune encephalomyelitis (EAE)]. We observed that double pathology was associated with a significantly earlier onset of disease, a slight increase in the neurological score, an increase in the number of infiltrating cells, and enhanced axonal degeneration compared with wild-type mice and the respective, single mutant controls. Double-mutant PLPtg/PD-1(-/-) mice showed an increased production of interferon-gamma by CNS immune cells at the peak of disease. Neither PD-1 deficiency nor oligodendropathy led to detectable spread of antigenic MHC class I- or class II-restricted epitopes during EAE. However, absence of PD-1 clearly increased the propensity of T lymphocytes to expand, and the number of clonal expansions reliably reflected the severity of the EAE disease course. Our data show that the interplay between immune dysregulation and myelinopathy results in a stable exacerbation of actively induced autoimmune CNS inflammation, suggesting that the combination of several pathological issues contributes significantly to disease susceptibility or relapses in human disease.

• PD-1 regulates neural damage in oligodendroglia-induced inflammation.

  Authors: Antje Kroner, Nicholas Schwab, Chi Wang Ip, Christoph Leder, Klaus-Armin Nave, Mathias Mäurer, Heinz Wiendl, Rudolf Martini

  PLoS ONE. 02/2009; 4(2):e4405.

  We investigated the impact of immune regulatory mechanisms involved in the modulation of the recently presented, CD8+ lymphocyte mediated immune response in a mouse model of oligodendropathy-inducedWe investigated the impact of immune regulatory mechanisms involved in the modulation of the recently presented, CD8+ lymphocyte mediated immune response in a mouse model of oligodendropathy-induced inflammation (PLPtg-mutants). The focus was on the role of the co-inhibitory molecule PD-1, a CD28-related receptor expressed on activated T- and B-lymphocytes associated with immune homeostasis and autoimmunity. PLPtg/PD-1-deficient double mutants and the corresponding bone marrow chimeras were generated and analysed using immunohistochemistry, light- and electron microscopy, with particular emphasis on immune-cell number and neural damage. In addition, the immune cells in both the CNS and the peripheral immune system were investigated by IFN-gamma elispot assays and spectratype analysis. We found that mice with combined pathology exhibited significantly increased numbers of CD4+ and CD8+ T-lymphocytes in the CNS. Lack of PD-1 substantially aggravated the pathological phenotype of the PLPtg mutants compared to genuine PLPtg mutants, whereas the PD-1 deletion alone did not cause alterations in the CNS. CNS T-lymphocytes in PLPtg/PD-1-/- double mutants exhibited massive clonal expansions. Furthermore, PD-1 deficiency was associated with a significantly higher propensity of CNS but not peripheral CD8+ T-cells to secrete proinflammatory cytokines. PD-1 could be identified as a crucial player of tissue homeostasis and immune-mediated damage in a model of oligodendropathy-induced inflammation. Alterations of this regulatory pathway lead to overt neuroinflammation of high pathogenetic impact. Our finding may have implications for understanding the mechanisms leading to the high clinical variability of polygenic or even monogenic disorders of the nervous system.

• The alpha-chemokine CXCL14 is up-regulated in the sciatic nerve of a mouse model of Charcot-Marie-Tooth disease type 1A and alters myelin gene expression in cultured Schwann cells.

  Authors: Elena M Barbaria, Bianca Kohl, Bettina A Buhren, Kerstin Hasenpusch-Theil, Fabian Kruse, Patrick Küry, Rudolf Martini, Hans Werner Müller

  Neurobiology of disease. 01/2009;

  At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model,At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model, which mimics the human CMT1A disorder. We observed that myelin gene expression in the sciatic nerve of the C61 mouse was up-regulated at postnatal day 4 to 7 (P4-P7). When investigating the morphology of peripheral nerves in C61 and wildtype mice at early stages of postnatal development, hypermyelination could be detected in the femoral quadriceps and sciatic nerve of transgenic animals at postnatal day 7 (P7). In order to identify genes, other than Pmp22, that are modulated in sciatic nerve of P7 transgenic mice, we applied microarray technology. Amongst the regulated genes, the gene encoding the alpha-chemokine CXCL14 was most prominently up-regulated. We report that Cxcl14 was expressed exclusively by Schwann cells of the sciatic nerve, as well as by cultured Schwann cells triggered to differentiate. Furthermore, in cultured Schwann cells CXCL14 modulated the expression of myelin genes and altered cell proliferation. Our findings demonstrate that early overexpression of PMP22, in a mouse model of CMT1A, results in a strong up-regulation of CXCL14, which seems to play a novel regulatory role in Schwann cell differentiation.

• Interactions between Schwann cells and macrophages in injury and inherited demyelinating disease.

  Authors: Rudolf Martini, Stefan Fischer, Rubèn López-Vales, Samuel David

  Glia. 10/2008; 56(14):1566-1577.

  In this article we first discuss the factors that regulate macrophage recruitment, activation, and myelin phagocytosis during Wallerian degeneration and some of the factors involved in theIn this article we first discuss the factors that regulate macrophage recruitment, activation, and myelin phagocytosis during Wallerian degeneration and some of the factors involved in the termination of inflammation at the end of the period of Wallerian degeneration after peripheral nerve injuries. In particular, we deal with the early events that trigger chemokine and cytokine expression; the role of phospholipase A(2) in initiating the breakdown of compact myelin, and chemokine, cytokine expression; and the role of MCP-1, MIP-1alpha, and IL-1beta in macrophage recruitment and myelin phagocytosis. We also discuss how inflammation may be switched off and the recently identified role of the Nogo receptor on activated macrophages in the clearance of these cells from the injured nerve. In the second half of the article we focus on the role of certain Schwann cell borne cytokines and chemokines, such as M-CSF and MCP-1 as well as intracellular signaling that regulate their expression in animal models of inherited demyelinating disease. Additionally, we present the preservation of sensory nerves fibers from macrophage attack in these animal models as a challenging paradigm for the development of putative treatment approaches. Finally, we also discuss the similarities and differences in these Schwann cell-macrophage responses in injury-induced Wallerian degeneration and inherited demyelinating diseases. Knowledge of the molecular mechanisms underlying Schwann cell-macrophage interaction under pathological conditions is an important prerequisite to develop effective treatment strategies for various peripheral nerve disorders. (c) 2008 Wiley-Liss, Inc.

• Increase of MCP-1 (CCL2) in myelin mutant Schwann cells is mediated by MEK-ERK signaling pathway.

  Authors: Stefan Fischer, Andreas Weishaupt, Jakob Troppmair, Rudolf Martini

  Glia. 07/2008; 56(8):836-43.

  Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation ofMacrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation of the Schwann cell-derived chemokine MCP-1 (CCL2) is a pathologically relevant mechanism for macrophage activation in mice heterozygously deficient for the myelin component P0 (P0+/-), we posed the question of the intracellular signaling cascade involved. By using western blot analysis of peripheral nerve lysates the MAP-kinases extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP kinase/ERK kinase 1/2 (MEK1/2) showed an early and constantly increasing activation in P0 mutants. Furthermore, in nerve fibers from the P0+/- mutants, Schwann cell nuclei were much more often positive for phosphorylated ERK1/2 than in nerve fibers from wild type mice. In vitro experiments using the MEK1/2-inhibitor CI-1040 decreased ERK1/2-phosphorylation and MCP-1 expression in a Schwann cell-derived cell line. Finally, systemic application of CI-1040 lead to a decreased ERK1/2-phosphorylation and substantially reduced MCP-1-production in peripheral nerves of P0+/- mutant mice. Our study identifies MEK1/2-ERK1/2 signaling as an important intracellular pathway that connects the Schwann cell mutation with the activation of pathogenetically relevant macrophages in the peripheral nerves. These findings may have important implications for the treatment of inherited peripheral neuropathies in humans.

• Origin of CD11b+ macrophage-like cells in the CNS of PLP-overexpressing mice: Low influx of haematogenous macrophages and unchanged blood-brain-barrier in the optic nerve.

  Authors: Chi Wang Ip, Bianca Kohl, Christoph Kleinschnitz, Bernhard Reuss, Klaus-Armin Nave, Antje Kroner, Rudolf Martini

  Molecular and cellular neurosciences. 06/2008;

  We have recently reported that overexpression of proteolipid protein in oligodendrocytes leads to a pathologically relevant increase of both CD8+ T-lymphocytes and CD11b+ cells in the CNS. We nowWe have recently reported that overexpression of proteolipid protein in oligodendrocytes leads to a pathologically relevant increase of both CD8+ T-lymphocytes and CD11b+ cells in the CNS. We now focussed on the origin of the CD11b+ cells in the optic nerve, a well established structure for the analysis of the mutant, using bone marrow chimeric mice. Although there is an age-related increase in CD11b+ cells in the myelinated part of the optic nerve of the mutants, the percentage of infiltrating cells was not increased, but enhanced proliferation was detectable. In the non-myelinated optic nerve head, the rate of infiltrating CD11b+ cells and albumin extravasation was high in both genotypes. However, albumin extravasation was also high in the rostral myelinated part, where CD11b+ cell influx was low. Our study demonstrates an intrinsic origin of CD11b+ cells in the presence of an unchanged blood-brain-barrier in a CNS myelin mutant.

• Monocyte chemoattractant protein-1 is a pathogenic component in a model for a hereditary peripheral neuropathy.

  Authors: Stefan Fischer, Christoph Kleinschnitz, Marcus Müller, Igor Kobsar, Chi Wang Ip, Barrett Rollins, Rudolf Martini

  Molecular and cellular neurosciences. 03/2008; 37(2):359-66.

  Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in models for inherited demyelinating neuropathies. It is presently unknown which factorsMacrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in models for inherited demyelinating neuropathies. It is presently unknown which factors link the Schwann cell-based myelin mutation to the activation of endoneurial macrophages. Here we identified the chemokine monocyte chemoattractant protein-1 (MCP-1) as a first and crucial factor upregulated in Schwann cells of mice heterozygously deficient for the myelin protein zero. The chemokine could be identified as an important mediator of macrophage immigration into peripheral nerves. Furthermore, a 50% reduction of chemokine expression by crossbreeding with MCP-1-deficient mice reduced the increase in macrophage numbers in the mutant nerves and lead to a robust amelioration of pathology. Surprisingly, the complete absence of MCP-1 aggravated the disease. Our findings show that reducing but not eliminating chemokine expression can rescue genetically caused demyelination that may be an interesting target in treating demyelinating diseases of the peripheral nervous system.

• Visualization of degenerating axons in a dysmyelinating mouse mutant with axonal loss.

  Authors: Birgit Ey, Igor Kobsar, Heinrich Blazyca, Antje Kroner, Rudolf Martini

  Molecular and cellular neurosciences. 06/2007; 35(1):153-60.

  Mice homozygously deficient for the myelin component P0 show loss of axons in peripheral nerves. In order to investigate the morphological characteristics of degenerating axons, we crossbred theMice homozygously deficient for the myelin component P0 show loss of axons in peripheral nerves. In order to investigate the morphological characteristics of degenerating axons, we crossbred the myelin mutants with a transgenic mouse line expressing yellow fluorescent protein (YFP) in a small proportion of neurons. Peripheral nerves of the double mutants were prepared into small fiber bundles and investigated by fluorescence microscopy. We could identify the tips of degenerating axon as bulb-like structures. Additionally, by electron microscopy, these structures were characterized as axoplasmic extensions containing numerous membraneous compartments. By immunoelectron microscopy, the degenerating end bulbs were in contact with ensheathing Schwann cells that contained YFP-immunoreactivity possibly reflecting phagocytosis of axon material by these cells. Immunohistochemistry using antibodies against macrophages revealed that YFP-positive bulbs, but also other axonal swellings, were often associated with macrophages supporting our previous findings that myelin-related axonal loss is partially mediated by these cells.

• Sialoadhesin deficiency ameliorates myelin degeneration and axonopathic changes in the CNS of PLP overexpressing mice.

  Authors: Chi Wang Ip, Antje Kroner, Paul R Crocker, Klaus-Armin Nave, Rudolf Martini

  Neurobiology of disease. 02/2007; 25(1):105-11.

  PLP overexpressing mice display demyelination and axonopathic changes, accompanied by an elevation of CD8+ T-lymphocytes and CD11b+ macrophages in the CNS. By crossbreeding these mutants withPLP overexpressing mice display demyelination and axonopathic changes, accompanied by an elevation of CD8+ T-lymphocytes and CD11b+ macrophages in the CNS. By crossbreeding these mutants with RAG-1-deficient mice lacking mature lymphocytes, we could recently demonstrate a pathogenetic impact of the CD8+ cells. In the present study, we investigated the pathogenetic impact of CD11b+ macrophages by crossbreeding the myelin mutants with knockout mice deficient for the macrophage-restricted adhesion molecule sialoadhesin (Sn). In the wild-type mice, Sn is barely detectable on CD11b+ cells, whereas in the myelin mutants, almost all CD11b+ cells express Sn. In the double mutants, upregulation of CD8+ T-cells and CD11b+ macrophages is reduced and pathological alterations are ameliorated. These data indicate that in a primarily genetically caused myelin disorder of the CNS macrophages expressing Sn partially mediate pathogenesis. These findings may have substantial impact on treatment strategies for leukodystrophic disorders and some forms of multiple sclerosis.

• Macrophage colony stimulating factor is a crucial factor for the intrinsic macrophage response in mice heterozygously deficient for the myelin protein P0.

  Authors: Marcus Müller, Martin Berghoff, Igor Kobsar, Reinhard Kiefer, Rudolf Martini

  Experimental neurology. 02/2007; 203(1):55-62.

  Mouse mutants heterozygously deficient for the myelin protein P0 (P0+/-) resemble certain forms of human hereditary neuropathies. Endoneurial macrophages of intrinsic origin are intimately involvedMouse mutants heterozygously deficient for the myelin protein P0 (P0+/-) resemble certain forms of human hereditary neuropathies. Endoneurial macrophages of intrinsic origin are intimately involved in the pathogenesis of the demyelinating neuropathy in these mutants. We have previously shown that deficiency for macrophage colony stimulating factor (M-CSF) prevents an increase of the number of endoneurial macrophages and alleviates the mutants' demyelinating phenotype. The aim of this study was to investigate which population of endoneurial macrophages - long-term resident macrophages or recently infiltrated macrophages - is affected by M-CSF deficiency. For this purpose, we generated bone marrow chimeric mice by transplanting GFP+ bone marrow into P0 mutants (P0+/-) and P0 mutants that lack M-CSF (P0+/- mcsf-op). This enabled us to discriminate recently infiltrated short-term resident GFP+ macrophages from long-term resident GFP- macrophages. Three months after bone marrow transplantation, P0+/- mice expressing M-CSF showed a substantial upregulation and activation of both GFP- and GFP+ macrophages in femoral nerves when compared to P0+/+ mice. In contrast, in P0+/- mcsf-op mutants, both GFP- and GFP+ macrophages did not substantially increase. Only small numbers of GFP+ but no GFP- macrophages were activated and phagocytosed myelin in chimeric P0+/- mcsf-op mutants, possibly reflecting recent activation outside the endoneurium before entering the nerve. Our findings demonstrate that M-CSF is crucial for the activation, in situ increase and myelin phagocytosis of both long-term and short-term resident endoneurial macrophages in P0+/- myelin mutants. M-CSF is, therefore, considered as a target candidate for therapeutic strategies to treat human demyelinating neuropathies.

• Immune cells contribute to myelin degeneration and axonopathic changes in mice overexpressing proteolipid protein in oligodendrocytes.

  Authors: Chi Wang Ip, Antje Kroner, Martin Bendszus, Christoph Leder, Igor Kobsar, Stefan Fischer, Heinz Wiendl, Klaus-Armin Nave, Rudolf Martini

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 09/2006; 26(31):8206-16.

  Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in whiteOverexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8+ T-lymphocytes and CD11b+ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8+ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-I+ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T- and B-lymphocytes led to a reduction of the number of CD11b+ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8+ versus CD4+ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4+) or CD4-negative (CD8+) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8+ mice, suggesting that the CD8+ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.

• Heterozygous P0 deficiency protects mice from vincristine-induced polyneuropathy.

  Authors: Nurcan Uçeyler, Igor Kobsar, Lydia Biko, Jochen Ulzheimer, S Rock Levinson, Rudolf Martini, Claudia Sommer

  Journal of neuroscience research. 08/2006; 84(1):37-46.

  Patients with hereditary neuropathies are more susceptible to vincristine (VIN)-induced neuropathy than patients without this comorbidity. The heterozygous P0(+/-) mouse is an animal model of aPatients with hereditary neuropathies are more susceptible to vincristine (VIN)-induced neuropathy than patients without this comorbidity. The heterozygous P0(+/-) mouse is an animal model of a distinct form of inherited neuropathies. These mice produce only 50% of the major myelin protein protein zero (P0) and display signs of demyelination in motor nerves at 4 months of age. Here we investigated the development of neuropathic signs in P0(+/-) and wild-type (wt) mice after VIN treatment. Neuropathy was induced by daily intraperitoneal injections of VIN (0.5 mg/kg body weight) over 10 days. Behavioral and electrophysiological tests were performed at regular time points. Wt mice developed significant hypersensitivity to heat and mechanical stimuli between days 7 and 38 after the first VIN injection. Surprisingly, P0(+/-) mice did not show sensory or motor signs of neuropathy over the whole testing period. Immunohistochemical analysis showed an increase in macrophage numbers in sciatic nerve sections of wt mice after VIN, whereas P0(+/-) mice had higher baseline levels of macrophages without changes after VIN treatment. Semithin sections revealed a decrease in the number of small-diameter myelinated fibers in the sciatic nerves of wt mice after VIN application, whereas P0(+/-) mice had higher baseline values of this fiber subtype that did not change under treatment. Dorsal root ganglion neurons of both genotypes showed an up-regulation of voltage-gated sodium channel immunoreactivity after VIN application without differences between the genotypes. Thus, the P0(+/-) phenotype seems to be protected against VIN-induced neuropathy. The mechanism of this neuroprotection remains elusive.

• Attenuated demyelination in the absence of the macrophage-restricted adhesion molecule sialoadhesin (Siglec-1) in mice heterozygously deficient in P0.

  Authors: Igor Kobsar, Cornelia Oetke, Antje Kroner, Carsten Wessig, Paul Crocker, Rudolf Martini

  Molecular and cellular neurosciences. 05/2006; 31(4):685-91.

  Mouse mutants heterozygously deficient for the myelin component P0 mimic some forms of inherited neuropathies in humans. We have previously shown that both T lymphocytes and macrophages contribute toMouse mutants heterozygously deficient for the myelin component P0 mimic some forms of inherited neuropathies in humans. We have previously shown that both T lymphocytes and macrophages contribute to the demyelinating neuropathy. Both cell types appear to influence each other mutually, i.e., impaired T lymphocyte development in RAG-1-deficient P0 mutants leads to decreased macrophage numbers and retarded macrophage activation causes reduced T lymphocyte numbers in the peripheral nerves of P0(+/-) mice. In the present study, we investigated the possible role of the macrophage-restricted sialic acid-binding Ig-like lectin sialoadhesin (Sn, Siglec-1) in the pathogenesis of inherited demyelination in P0(+/-) mice. We found that most peripheral nerve macrophages express Sn in the mutants. Myelin mutants devoid of Sn show reduced numbers of CD8+ T lymphocytes and macrophages in peripheral nerves and less severe demyelination, resulting in improved nerve conduction properties. Our findings are potentially important in the development of future treatment strategies for inherited demyelinating neuropathies.

• Role of immune cells in animal models for inherited peripheral neuropathies.

  Authors: Chi Wang Ip, Antje Kroner, Stefan Fischer, Martin Berghoff, Igor Kobsar, Mathias Mäurer, Rudolf Martini

  Neuromolecular medicine. 02/2006; 8(1-2):175-90.

  Mice expressing half of the normal dose of protein zero (P0+/- mice) or completely deficient gap-junction protein connexin 32 -/- mice mimic demyelinating forms of inherited neuropathies, such asMice expressing half of the normal dose of protein zero (P0+/- mice) or completely deficient gap-junction protein connexin 32 -/- mice mimic demyelinating forms of inherited neuropathies, such as Charcot-Marie-Tooth (CMT) neuropathies type 1B and CMT type 1X, respectively. In both models, an almost normal myelin formation is observed during the first months of life, followed by a slowly progressing demyelinating neuropathy. In both models, there is a substantial increase of CD8+ T-lymphocytes and macrophages within the demyelinating nerves. Recently, this has also been observed in mice mildly overexpressing human peripheral myelin protein 22 kD mimicking the most common form of CMT, CMT type 1A. In all demyelinating models, the macrophages show close contacts with intact myelin sheaths or demyelinated axons, suggesting an active role of these cells in myelin degeneration. Additionally, fibroblast-like cells contact macrophages, suggesting a functional role of fibroblast-like cells in macrophage activation. By cross-breeding P0+/- and gap-junction protein connexin 32-/- mice with immunodeficient recombination activating gene-1-deficient mutants, a substantial alleviation of the demyelinating phenotype was observed. Similarly, cross-breeding of P0+/- mice with mutants with a defect in macrophage activation led to an alleviated phenotype as well. These findings demonstrate that the immune system is involved in the pathogenesis of demyelinating neuropathies. In contrast, in P0-/- mice, which display a compromised myelin compaction and axonal loss from onset, immune cells appear to have a neuroprotective effect because cross-breeding with recombination activating gene-1 mutants leads to an aggravation of axonopathic changes. In the present review, we discuss the influence of the immune system on inherited de- and dysmyelination regarding disease mechanisms and possible clinical implications.