Klaus V. Toyka

CNTF is a major protective factor in demyelinating CNS disease: A neurotrophic cytokine as modulator in neuroinflammation

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). So far, immunological mechanisms responsible for demyelination have been the focus of interest. However, mechanisms regulating axon maintenance as well as glial precursor-cell proliferation and oligodendrocyte survival might also influence disease outcome. The cytokine ciliary neurotrophic factor (CNTF), which was originally identified as a survival factor for isolated neurons, promotes differentiation, maturation and survival of oligodendrocytes. To investigate the role of endogenous CNTF in inflammatory demyelinating disease, we studied myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in CNTF-deficient and wild-type C57BL/6 mice. Disease was more severe in CNTF-deficient mice and recovery was poor, with a 60% decrease in the number of proliferating oligodendrocyte precursor cells (OPCs) and a more than 50% increase in the rate of oligodendrocyte apoptosis. In addition, vacuolar dystrophy of myelin and axonal damage were more severe in CNTF-deficient mice. These specific pathological features could be prevented by treatment with an antiserum against tumor necrosis factor-α, suggesting that endogenous CNTF may counterbalance this effect of TNF-α (ref. 7). Here we identify a factor that modulates, in an inflammatory environment, glial cell survival and is an outcome determinant of EAE.

Mice deficient for the myelin-associated glycoprotein show subtle abnormalities in myelin

Using homologous recombination in embryonic stem cells, we have generated mice with a null mutation in the gene encoding the myelin-associated glycoprotein (MAG), a recognition molecule implicated in myelin formation. MAG-deficient mice appeared normal in motor coordination and spatial learning tasks. Normal myelin structure and nerve conduction in the PNS, with N-CAM overexpression at sites normally expressing MAG, suggested compensatory mechanisms. In the CNS, the onset of myelination was delayed, and subtle morphological abnormalities were detected in that the content of oligodendrocyte cytoplasm at the inner aspect of most myelin sheaths was reduced and that some axons were surrounded by two or more myelin sheaths. These observations suggest that MAG participates in the formation of the periaxonal cytoplasmic collar of oligodendrocytes and in the recognition between oligodendrocyte processes and axons.

Animal models for autoimmune demyelinating disorders of the nervous system

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that takes a relapsing-remitting or a progressive course (reviewed in Refs 1,2). Its counterpart in the peripheral nervous system (PNS) is chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) (reviewed in Ref. 3). In addition, there are acute, monophasic disorders, such as the inflammatory demyelinating polyradiculoneuropathy termed Guillain-Barré syndrome (GBS) in the PNS, and acute disseminated encephalomyelitis (ADEM) in the CNS. Both MS and GBS are heterogeneous syndromes. In MS different exogenous assaults together with genetic factors can result in a disease course that finally fulfils the diagnostic criteria. In both diseases, axonal damage can add to a primarily demyelinating lesion and cause permanent neurological deficits. No single animal model exists that mimics all the features of human demyelinating diseases; rather, the available models reflect specific facets. Here, we focus on experimental autoimmune encephalomyelitis (EAE) and neuritis (EAN) as models in rat and mouse strains, and discuss their distinct histopathology and the roles played by different autoantigens.

Axonal prion protein is required for peripheral myelin maintenance

The integrity of peripheral nerves relies on communication between axons and Schwann cells. The axonal signals that ensure myelin maintenance are distinct from those that direct myelination and are largely unknown. Here we show that ablation of the prion protein PrPC triggers a chronic demyelinating polyneuropathy (CDP) in four independently targeted mouse strains. Ablation of the neighboring Prnd locus, or inbreeding to four distinct mouse strains, did not modulate the CDP. CDP was triggered by depletion of PrPC specifically in neurons, but not in Schwann cells, and was suppressed by PrPC expression restricted to neurons but not to Schwann cells. CDP was prevented by PrPC variants that undergo proteolytic amino-proximal cleavage, but not by variants that are nonpermissive for cleavage, including secreted PrPC lacking its glycolipid membrane anchor. These results indicate that neuronal expression and regulated proteolysis of PrPC are essential for myelin maintenance.

A phase I/II trial of recombinant methionyl human brain derived neurotrophic factor administered by intrathecal infusion to patients with amyotrophic lateral sclerosis.

BACKGROUND: Brain derived neurotrophic factor (BDNF) is a potent survival factor for motoneurons. This study investigated the safety and tolerability of recombinant methionyl ( r -metHuBDNF) infused intrathecally by means of an implanted pump in patients with ALS. METHODS: Twenty-five probable or definite ALS were treated with either r -metHuBDNF (25, 60, 150, 400 or 1000 mug/day) or placebo in a 12- week, randomized, double-blinded, sequential, dose-escalation study. Test treatment was interrupted by a washout period from days 11 to 25 to allow the evaluation of laboratory safety measures. In each dose cohort four patients received r -metHuBDNF and one received placebo. On completion of the double-blind period of the study all patients continued to receive r -metHuBDNF in an open-label extension for up to 60 weeks. Lumbar cerebrospinal fluid (CSF) samples were taken periodically from all patients for the measurement of r -metHuBDNF levels and in a minority of patients these were supplemented by cisternal samples. RESULTS: Within days after the initiation of infusion the majority of patients receiving r -metHuBDNF reported mild sensory symptoms, including paraesthesias or a human BDNF sense of warmth, which were usually confined to the lower limbs and were frequently exacerbated by neck flexion. In most instances these symptoms patients with decreased or even disappeared over several weeks. Sleep disturbance, dry mouth, agitation and other behavioural effects were encountered at higher doses (>150 mug/day) and necessitated dose reductions. The spinal CSF levels of r -metHuBDNF were directly related to dose, with a lumbar to cervical ratio of approximately 4:1. CONCLUSIONS: The intrathecal delivery of r -metHuBDNF in doses of up to 150 mug/day was well tolerated and appears feasible. The reversible CNS effects with higher dose indicate that BDNF can be delivered cranially against CSF flow. The small number of patients and the design of the study did not permit conclusions to be drawn about the efficacy of the treatment. (ALS 2000; 1:201-206)

Myasthenia gravis Long‐term correlation of binding and bungarotoxin blocking antibodies against acetylcholine receptors with changes in disease severity

Sixty patients with myasthenia gravis were examined prospectively by measuring serial titers of antibodies against human acetylcholine receptor, and these were correlated with a quantitative clinical score. Serial titers of antibodies detected by the standard immunoprecipitation assay (binding antibodies) correlated with the clinical score in most patients. Antibodies blocking the binding of α-bungarotoxin to receptors (blocking antibodies) were detected in 29 patients. Serial blocking antibody titers correlated with changes in muscle weakness less often than binding antibody titers. Titers of both classes of antibodies often followed a divergent course, suggesting that the autoimmune B-cell clones that formed these classes of antibodies may have been activated asynchronously.

Production of nitrite by neonatal rat microglial cells/ brain macrophages

Microglial cells/brain macrophages from neonatal rats were examined for their capacity to generate nitrite, a product of the NO pathway. Upon incubation with bacterial lipopolysaccharide (LPS) or rat interferon-gamma (IFN-gamma), cells from the microglia-enriched fraction released measurable amounts of nitrite into the supernatant within 24-48 hr. The production of nitrite was dependent on the cell number and the dose of IFN-gamma and LPS. It could be inhibited by NG-monomethylarginine. We conclude that activated microglial cells can secrete nitrite. Stimulation of the NO pathway in microglial cells may be relevant to the pathogenesis of inflammatory and autoimmune demyelinating diseases of the brain.

A role for BDNF in mechanosensation

Brain-derived neurotrophic factor (BDNF) is a survival factor for certain sensory neurons during development. Using electrophysiology in BDNF-deficient mice, we show here that slowly adapting mechanoreceptors (SAM), but not other types of cutaneous afferents, require BDNF in postnatal life for normal mechanotransduction. Neurons lacking BDNF did not die, but instead showed a profound and specific reduction in their mechanical sensitivity, which was quantitatively the same in BDNF -/- and BDNF +/- animals. Postnatal treatment of BDNF +/- mice with recombinant BDNF completely rescued the mechanosensitivity deficit. Therefore BDNF is important for regulating SAM mechanosensitivity, independent of any survival-promoting function.

Paraneoplastic stiff-person syndrome: passive transfer to rats by means of IgG antibodies to amphiphysin

BACKGROUND Stiff-person syndrome (SPS) with antibodies to amphiphysin is a paraneoplastic disorder of the central nervous system with a putative autoimmune pathogenesis. Proof of a causal role of the antibodies is still lacking for this and all other antibody-associated paraneoplastic syndromes of the central nervous system. METHODS We obtained the plasma filtrate of a patient with breast cancer and SPS that responded to therapeutic plasmapheresis. The purified IgG fraction included high-titre antibodies to the synaptic protein amphiphysin. In a cotransfer design, this IgG fraction was injected intraperitoneally into female Lewis rats that had received encephalitogenic T-helper (Th) lymphocytes specific for myelin basic protein, to induce an immune-mediated leaky blood-brain barrier. The rats were followed up with behavioural tests, video photography, and electromyography. FINDINGS The injection of the IgG fraction including antibodies to amphiphysin resulted in a dose-dependent stiffness with spasms resembling human SPS. Control IgG injected into rats that had received the same encephalitogenic Th cells had no effect. IgG binding was demonstrated in the central nervous system of rats that showed signs of the disorder. INTERPRETATION These experiments support the hypothesis of a pathogenetic role of antibodies to amphiphysin, thus adding paraneoplastic SPS to the group of antibody-mediated autoimmune disorders. RELEVANCE TO PRACTICE These findings provide a strong argument for a direct pathogenetic role of anti-amphiphysin in this type of SPS and support therapeutic attempts to eliminate these autoantibodies by plasmapheresis. The experimental approach used could help to elucidate the role of autoantibodies in other paraneoplastic syndromes, such as SPS with antibodies to glutamic acid decarboxylase, and others including anti-Hu-associated subacute cerebellar degeneration and limbic encephalitis.

Etanercept reduces hyperalgesia in experimental painful neuropathy

Abstract  Etanercept, a recombinant tumor necrosis factor receptor (p75)‐Fc fusion protein competitively inhibits tumor necrosis factor‐alpha (TNF). Etanercept has been successfully used in patients with rheumatoid arthritis, where it reduces pain and inflammation. Because locally produced proinflammatory cytokines play a role in pain after nerve injury, we investigated whether etanercept can reduce pain and hyperalgesia in an animal model of painful neuropathy, the chronic constriction injury of the sciatic nerve. C57BL/6 mice received etanercept or sham treatment by local near‐nerve injection to the injured nerve or by systemic application. Treatment with etanercept reduced thermal hyperalgesia and mechanical allodynia significantly in both modes of application. The effect of etanercept was present in animals that were treated from the time of surgery and in those that were treated from day 6, when hyperalgesia was already present. These results suggest the potential of etanercept as a treatment option for patients with neuropathic pain.