Immune-mediated inflammatory polyneuropathy overlapping Charcot-Marie-Tooth 1B.

Charcot Marie Tooth (CMT) due to myelin protein zero (MPZ) mutations, may cause a wide variation of phenotypes, depending on the localization of the mutation within the gene. Among the most common phenotypes are: an infantile onset disease with extremely slow nerve conduction velocities (CMT1B) and an adult onset phenotype with nerve velocities in the axonal range (CMT2I). We reported a patient with CMT1B (MPZ p.Ser63del mutation) which developed an overlapping immune mediated polyradiculoneuropathy with recurrent episodes of quadriparesis and cranial nerve involvement. We observed reversible conduction block on serial neurophysiologic studies, non-uniform demyelination and good clinical response to prednisone and cyclophosphamide, as evidenced by objective functional recovery. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP)-like characteristics have not yet been described associated with a MPZ p.Ser63del mutation. This description adds evidence indicating that a defective structural myelin protein may predispose peripheral nerves to immune attacks.

Neuroinflammation in the pathogenesis of axonal Charcot-Marie-Tooth disease caused by lack of GDAP1.

Mutations in the GDAP1 mitochondrial outer membrane gene cause Charcot-Marie-Tooth (CMT) neuropathy. Reduction or absence of GDAP1 has been associated with abnormal changes in the mitochondrial morphology and dynamics, oxidative stress and changes in calcium homeostasis. Neuroinflammation has been described in rodent models of genetic demyelinating CMT neuropathies but not in CMT primarily associated with axonopathy. Inflammatory processes have also been related to mitochondrial changes and oxidative stress in central neurodegenerative disorders. Here we investigated the presence of neuroinflammation in the axonal neuropathy of the Gdap1-/- mice. We showed by transcriptome profile of spinal cord and the in vivo detection of activated phagocytes that the absence of GDAP1 is associated with upregulation of inflammatory pathways. We observed reactive gliosis in spinal cord with increase of the astroglia markers GFAP and S100B, and the microglia marker IBA1. Additionally, we found significant increase of inflammatory mediators such as TNF-α and pERK, and C1qa and C1qb proteins of the complement system. Importantly, we observed an increased expression of CD206 and CD86 as M2 and M1 microglia and macrophage response markers, respectively, in Gdap1-/- mice. These inflammatory changes were also associated with abnormal molecular changes in synapses. In summary, we demonstrate that inflammation in spinal cord and sciatic nerve, but not in brain and cerebellum, is part of the pathophysiology of axonal GDAP1-related CMT.

Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40-60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS.

Targeting the colony stimulating factor 1 receptor alleviates two forms of Charcot-Marie-Tooth disease in mice.

See Scherer (doi:10.1093/awv279) for a scientific commentary on this article.Charcot-Marie-Tooth type 1 neuropathies are inherited disorders of the peripheral nervous system caused by mutations in Schwann cell-related genes. Typically, no causative cure is presently available. Previous preclinical data of our group highlight the low grade, secondary inflammation common to distinct Charcot-Marie-Tooth type 1 neuropathies as a disease amplifier. In the current study, we have tested one of several available clinical agents targeting macrophages through its inhibition of the colony stimulating factor 1 receptor (CSF1R). We here show that in two distinct mouse models of Charcot-Marie-Tooth type 1 neuropathies, the systemic short- and long-term inhibition of CSF1R by oral administration leads to a robust decline in nerve macrophage numbers by ∼70% and substantial reduction of the typical histopathological and functional alterations. Interestingly, in a model for the dominant X-linked form of Charcot-Marie-Tooth type 1 neuropathy, the second most common form of the inherited neuropathies, macrophage ablation favours maintenance of axonal integrity and axonal resprouting, leading to preserved muscle innervation, increased muscle action potential amplitudes and muscle strengths in the range of wild-type mice. In another model mimicking a mild, demyelination-related Charcot-Marie-Tooth type 1 neuropathy caused by reduced P0 (MPZ) gene dosage, macrophage blockade causes an improved preservation of myelin, increased muscle action potential amplitudes, improved nerve conduction velocities and ameliorated muscle strength. These observations suggest that disease-amplifying macrophages can produce multiple adverse effects in the affected nerves which likely funnel down to common clinical features. Surprisingly, treatment of mouse models mimicking Charcot-Marie-Tooth type 1A neuropathy also caused macrophage blockade, but did not result in neuropathic or clinical improvements, most likely due to the late start of treatment of this early onset disease model. In summary, our study shows that targeting peripheral nerve macrophages by an orally administered inhibitor of CSF1R may offer a highly efficacious and safe treatment option for at least two distinct forms of the presently non-treatable Charcot-Marie-Tooth type 1 neuropathies.

Molecular triggers of neuroinflammation in mouse models of demyelinating diseases.

Myelinating cells wrap axons with multi-layered myelin sheaths for rapid impulse propagation. Dysfunctions of oligodendrocytes or Schwann cells are often associated with neuroinflammation, as observed in animal models of leukodystrophies and peripheral neuropathies, respectively. The neuroinflammatory response modulates the pathological changes, including demyelination and axonal injury, but also remyelination and repair. Here we discuss different immune mechanisms as well as factors released or exposed by myelinating glia in disease conditions. The spectrum of inflammatory mediators varies with different myelin disorders and has a major impact on the beneficial or detrimental role of immune cells in keeping nervous system integrity.

Thrombotic thrombocytopenic purpura with severe ADAMTS-13 deficiency in a patient with antiphospholipid antibodies and Charcot-Marie-Tooth disease.

A 26-year-old woman with a history of mild mental retardation, Charcot-Marie-Tooth disease (CMT) and idiopathic thrombocytopenic purpura developed severe thrombocytopenia with Coombs-negative hemolytic anemia. Magnetic resonance imaging revealed a fresh cerebral infarction in the left precentral gyrus. ADAMTS-13 deficiency caused by an inhibitor and anti-cardiolipin antibodies were detected in the blood. After treatment with prednisolone and fresh frozen plasma, ADAMTS-13 activity was normalized, the ADAMTS-13 inhibitor had disappeared and the thrombocytopenia with a bleeding tendency was improved. To our knowledge, this is the first case of thrombotic thrombocytopenic purpura caused by ADAMTS-13 deficiency associated with antiphospholipid antibodies and CMT.

Isolated central nervous system relapse in an adolescent with acute myelomonocytic leukemia, Charcot Marie Tooth syndrome, and paraneoplastic autoantibody.

A 17-year-old boy, with acute myelomonocytic leukemia and inversion 16(p13q22) developed polyneuropathy and isolated central nervous system relapse. Scoliosis and high-arched feet suggested a diagnosis of Charcot Marie Tooth (CMT) syndrome and genetic testing confirmed duplication at the PMP22 locus at chromosome 17p11.12. No mutation was found in another CMT gene, the CMT C1 LITAF locus at 16p13.2, to suggest that this association is anything more than chance. Titres to VGKC, a paraneoplastic autoantibody, were elevated, suggesting an additional mechanism for the polyneuropathy. This case extends the clinical spectrum of cancer with CMT, and of paraneoplastic disorders.

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

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, 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.

Genetically determined neuropathy (CMT 1A) accompanied by immune dysfunction: a case report.

Peripheral Myelin Protein 22 (PMP22) is mostly expressed in Schwann cells where it is essential in the compaction of myelin. The duplication of the PMP22 gene results in a hereditary demyelinating neuropathy of the Charcot-Marie-Tooth type 1A (CMT1A). So far there are only a few case reports suggesting that dysimmune mechanisms may take part in the pathophysiology of this disease. We describe three siblings carrying the duplication of the PMP22 gene, with a significant reduction of serum immunoglobulin G levels in all three cases and sural nerve vasculitis in the two women, which supports the proposition, that immune dysfunction may accompany this disease in some cases.

Immune effects of mesenchymal stem cells: implications for Charcot-Marie-Tooth disease.

Mesenchymal stem cell (MSC) therapy is the most clinically advanced form of cell therapy, second to hematopoietic stem cell transplants. To date, MSC have been used for immune modulation in conditions such as Graft Versus Host Disease (GVHD) and Crohn's Disease, for which Phase III clinical trials are currently in progress. Here, we review the immunological properties of MSC and make a case for their use in treatment of Charcot-Marie-Tooth disease type 1 (CMT1), a group of inherited peripheral neuropathies. CMT1 is characterized by demyelination and aberrant immune activation making this condition an ideal target for exploration of MSC therapy, given the ability of these cells to promote sheath regeneration as well as suppress inflammation. Studies supporting this hypothesis will be presented and placed into the context of other cell-based approaches that are theoretically feasible. Given that MSCs selectively home to areas of inflammation, as well as exert effects in an allogeneic manner, the possibility of an "off the shelf" therapy for CMT1 will be discussed.

[Acute relapse in Charcot-Marie-Tooth 1B neuropathy: can protein P0 behave like an autoantigen?]. / Aggravation aiguë régressive d'une maladie de Charcot-Marie-Tooth de type 1B: la Protéine P0 peut-elle agir comme un auto-antigène?

INTRODUCTION: The natural history of Charcot-Marie-Tooth neuropathy is marked by accentuated motor and sensitive deficits suggestive of acute polyradiculoneuritis or, more generally, chronic inflammatory demyelinizing polyneuropathy. OBSERVATION: A 41-year-old woman, with Charcot-Marie-Tooth (CMT) 1B neuropathy associated with a P0 gene mutation, developed several episodes of ataxia which resolved after intravenous administration of IgG or corticosteroids. CONCLUSION: The sudden increase of a motor or sensitive deficit in this patient with CMT type I led to two hypotheses: chance association between an inherited and an inflammatory neuropathy, or a dysimmune inflammatory reaction, the mutated protein acting like an autoantigen. These two hypotheses are discussed.

Immune-mediated components of hereditary demyelinating neuropathies: lessons from animal models and patients.

Most demyelinating forms of Charcot-Marie-Tooth type 1 (CMT1) neuropathy are slowly progressive and do not respond to anti-inflammatory treatment. In nerve biopsy samples, overt lymphocytic infiltration is absent, but pathological features typical of macrophage-related demyelination have been reported. In mouse models of CMT1, demyelination was substantially reduced when the mutants were backcrossed into an immunodeficient genetic background. A few individual patients with CMT1 respond to anti-inflammatory treatment; however, unlike most patients with CMT1, these patients show accelerated worsening of symptoms, inflammatory infiltrates in nerve biopsies, and clinical features resembling chronic inflammatory demyelinating polyneuropathy as well as CMT1. We conclude that in patients with typical CMT1 and in animal models, a cryptic and mild inflammatory process not responsive to standard anti-inflammatory treatment fosters genetically mediated demyelination.

Inflammatory demyelination in a patient with CMT1A.

We report a case of Charcot-Marie-Tooth disease (CMT), with identified PMP22 gene duplication (CMT type 1A), and with evidence of an inflammatory demyelinating process superimposed on the course of the chronic genetic disease. Macrophage-associated demyelination was observed on the peripheral nerve biopsy. This observation supports some experimental data from the literature and shows that there may be a genetic susceptibility to inflammatory demyelinating processes in certain CMT kindreds.

Preserved myelin integrity and reduced axonopathy in connexin32-deficient mice lacking the recombination activating gene-1.

Mice heterozygously deficient for myelin protein zero (P0) mimicking human Charcot-Marie-Tooth (CMT) disease 1B show T-lymphocyte and macrophage upregulation in peripheral nerves, which aggravates and modulates the genetically mediated demyelinating neuropathy. In connexin32 (cx32)-deficient (cx32(def)) mice, which mimic the X-linked dominant form of CMT (CMTX), T-lymphocyte and macrophage numbers are also significantly elevated in peripheral nerves. To test the hypothesis that immune cells are indeed pathogenic in this model, we cross-bred cx32(def) mice with recombination activating gene-1 (RAG-1)-deficient mice, which lack mature T- and B-lymphocytes. In these immunoincompetent double mutants, the number of endoneurial macrophages was reduced. Furthermore, features indicative of myelin degeneration and axonopathic changes were mitigated in the RAG-1-deficient double mutants, whereas enlarged periaxonal Schwann cell collars, a hallmark specific for cx32-mutants, were not reduced. Since both cx32- and P0 deficiency lead to similar immunopathogenic processes, we conclude that immune-mediated demyelination may be a feature common to many CMT-like neuropathies independent of the genetic origin.

Role of immune cells in animal models for inherited neuropathies: facts and visions.

Mice heterozygously deficient in the peripheral myelin adhesion molecule P0 (P0+/- mice) are models for some forms of Charcot-Marie-Tooth (CMT) neuropathies. In addition to the characteristic hallmarks of demyelination, elevated numbers of CD8-positive T-lymphocytes and F4/80-positive macrophages are striking features in the nerves of these mice. These immune cells increase in number with age and progress of demyelination, suggesting that they might be functionally related to myelin damage. In order to investigate the pathogenetic role of lymphocytes, the myelin mutants were cross-bred with recombination activating gene 1 (RAG-1)-deficient mice, which lack mature T- and B-lymphocytes. The immunodeficient myelin mutants showed a less severe myelin degeneration. The beneficial effect of lymphocyte-deficiency was reversible, since demyelination worsened in immunodeficient myelin-mutants when reconstituted with bone marrow from wild-type mice. Ultrastructural analysis revealed macrophages in close apposition to myelin and demyelinated axons. We therefore cross-bred the P0+/- mice with spontaneous osteopetrotic (op) mutants deficient in the macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the corresponding double mutants the numbers of macrophages were not elevated in the peripheral nerves, and the demyelinating phenotype was less severe than in the genuine P0+/- mice, demonstrating that macrophages are also functionally involved in the pathogenesis of genetically mediated demyelination. We also examined other models for inherited neuropathies for a possible involvement of immune cells. We chose mice deficient in the gap junction component connexin 32, a model for the X-linked form of CMT. Similar to P0-deficient mice, T-lymphocytes and macrophages were elevated and macrophages showed a close apposition to degenerating myelin. We conclude that the involvement of T-lymphocytes and macrophages is a common pathogenetic feature in various forms of slowly progressive inherited neuropathies.

Immunological study of hereditary motor and sensory neuropathy type 1a (HMSN1a).

OBJECTIVES: Fifty three patients were studied to investigate whether autoimmune or inflammatory mechanisms could explain the phenotypic heterogeneity of patients with hereditary motor and sensory neuropathy type 1a (HMSN1a). METHODS: Serum samples were examined for antibodies to peripheral nerve myelin protein 22 (PMP22), ganglioside GM1 and cauda equina homogenate, and interleukin-6 (IL-6) and soluble tumour necrosis factor receptor 1 (sTNF R1) concentrations. Serological results were compared with those from patients with other neuropathies (ONPs, n=30) and with normal subjects (n=51). RESULTS: In the group as a whole, no relation emerged between clinical severity and any immune parameters. Immunohistochemical examination of four sural nerve biopsies did not show significant inflammatory infiltration. In a subset of 12 patients who experienced stepwise progression of disease, there was a trend towards a higher proportion having anti-PMP22 antibodies (33% v 15% of those with gradual disease progression, 3% ONPs, and no normal controls) and complement fixing antibodies to human cauda equina (25% v 5% with gradual progression, 8.6% ONPs, 3.9% normal controls, p=0.07). CONCLUSIONS: Patients with HMSN1a and a stepwise disease progression may have an inflammatory, autoimmune component superimposed on the genetic condition.

Expression of IL-17B in neurons and evaluation of its possible role in the chromosome 5q-linked form of Charcot-Marie-Tooth disease.

IL-17B is a recently identified homolog of IL-17. Northern analysis revealed that IL-17B mRNA is expressed at very high levels in spinal cord and at much lower and more variable levels in trachea, prostate, lung, small intestine, testes, adrenal, and pancreas. In developing mouse embryos IL-17B expression was first detected at day 11 and appeared to peak at day 15. In situ analysis of mouse spinal cord, dorsal root ganglia, and brain demonstrated that IL-17B mRNA is primarily expressed by the neurons. Immunohistochemical analysis of human spinal cord, dorsal root ganglia, cerebral cortex, cerebellum, and hippocampus demonstrated that IL-17B protein is primarily localized to the neuronal cell bodies and axons. Radiation hybrid mapping localized the IL-17B gene to a region on human chromosome 5q that is associated with a rare autosomal recessive form of Charcot-Marie-Tooth demyelinating disease. However, no changes were found in the coding regions, splice junctions, intron 1, or the 5' and 3' untranslated regions of IL-17B genes of patients affected with this disease.

Characterisation of autoantibodies to peripheral myelin protein 22 in patients with hereditary and acquired neuropathies.

To investigate the possibility that an autoimmune mechanism may play a role in the hereditary neuropathy Charcot-Marie-Tooth type 1A (CMT1A), sera were analysed by Western blot for anti-peripheral myelin protein 22 (PMP22) autoantibodies. These sera were compared with sera from patients with CMT type 2 (CMT2), acquired peripheral neuropathies such as chronic inflammatory demyelinating neuropathy (CIDP), anti-MAG IgM neuropathy, Miller-Fisher syndrome (MFS), diabetic neuropathy and with control blood donors. Anti-PMP22 positive sera were detected in 70% of patients with CMT1 and unexpectedly in 60% of patients with CMT2. Interestingly, 44% of the patients with other peripheral neuropathies and 23% of the apparently healthy controls showed also anti-PMP22 antibody reactivity. Immunohistochemical analysis of the human anti-PMP22 antisera on healthy sural nerve sections and on PMP22-expressing COS cells revealed that these sera did not recognise endogenous PMP22. Our results indicate that anti-PMP22 autoantibodies are found in sera of patients with different types of peripheral neuropathies, but their role in the pathogenesis of these diseases remains to be determined.

Expression of glial cell line-derived neurotrophic factor and GDNFR-alpha mRNAs in human peripheral neuropathies.

The steady-state mRNA levels of glial cell line-derived neurotrophic factor (GDNF), GDNFR-alpha and RET were examined in various human peripheral neuropathies to determine the relationship with myelinated fiber pathology, and T cell and macrophage invasions in the diseased nerves. GDNF and GDNFR-alpha mRNA levels were elevated to variable extent in the diseased nerves, although they were not specific to the type of diseases. The increase of GDNFR-alpha mRNA levels was correlated with the extent of the nerves with axonal pathology, and was proportional to the extent of invasion of the nerves by T cells and macrophages. The GDNF mRNA levels were not related to axonal, demyelinating pathology, or inflammatory cell invasions. RET mRNA expression was not detected in normal nor diseased nerves. The GDNF and GDNFR-alpha expression in the diseased human nerves is regulated by an underlying pathology-related process, and could play a role in peripheral nerve repair.