Jürgen Zielasek

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.

Preceding infections, immune factors, and outcome in Guillain–Barré syndrome

Objective: To test the hypothesis that different preceding infections influence the neurophysiologic classification and clinical features of Guillain–Barré syndrome (GBS). Methods: We tested pretreatment sera, 7 ± 3 (mean ± SD) days from onset, from 229 patients with GBS in a multicenter trial of plasma exchange and immunoglobulin, for serological markers of infection, adhesion molecules, and cytokine receptors, and compared these with neurophysiologic and clinical features. Results: Recent infection by Campylobacter jejuni was found in 53 patients (23%), cytomegalovirus in 19 (8%), and Epstein–Barr virus in four (2%). Patients with C. jejuni infection were more likely than others to have neurophysiologic criteria of axonal neuropathy or inexcitable nerves, antiganglioside GM1 antibodies, pure motor GBS, lower CSF protein, and worse outcome. Patients with cytomegalovirus infection were younger and more likely than others to have raised serum concentrations of molecules important in T lymphocyte activation and migration, soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble leukocyte selectin, and soluble interleukin-2 receptor (sIL-2R). Concentrations of sICAM-1 and soluble tumor necrosis factor receptor were higher in patients with inexcitable nerves than those with demyelinating neurophysiology. Logistic regression analysis showed death or inability to walk unaided at 48 weeks were associated with diarrhea, inexcitable nerves, severe arm weakness, age over 50, raised sIL-2R concentration and absence of immunoglobulin (Ig) M antiganglioside GM1 antibodies. Conclusions: Subtypes of GBS defined by preceding infections were only approximately associated with different patterns of clinical, neurophysiologic, and immunologic features. A single infectious agent caused more than one type of pathology in GBS, implying interaction with additional host factors. Most patients had no identified infection.

Molecular mechanisms of microglial activation

Microglial cells are brain macrophages which serve specific functions in the defense of the central nervous system (CNS) against microorganisms, the removal of tissue debris in neurodegenerative diseases or during normal development, and in autoimmune inflammatory disorders of the brain. In cultured microglial cells, several soluble inflammatory mediators such as cytokines and bacterial products like lipopolysaccharide (LPS) were demonstrated to induce a wide range of microglial activities, e.g. increased phagocytosis, chemotaxis, secretion of cytokines, activation of the respiratory burst and induction of nitric oxide synthase. Since heightened microglial activation was shown to play a role in the pathogenesis of experimental inflammatory CNS disorders, understanding the molecular mechanisms of microglial activation may lead to new treatment strategies for neurodegenerative disorders, multiple sclerosis and bacterial or viral infections of the nervous system.

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.

Administration of nitric oxide synthase inhibitors in experimental autoimmune neuritis and experimental autoimmune encephalomyelitis

The nitric oxide (NO) synthase pathway is activated during experimental autoimmune inflammation of the central nervous system, and administration of aminoguanidine, an inhibitor of the cytokine-inducible NO synthase (NOS), ameliorated the disease course of autoimmune encephalomyelitis in the SJL mouse. We studied the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune neuritis (EAN) and experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. NG-L-monomethyl-arginine (L-NMMA), a competitive inhibitor of NOS, partially suppressed T cell line-mediated EAN, but not myelin-induced EAN, myelin basic protein (MBP)-induced EAE, or T cell line-mediated EAE. Aminoguanidine (AG), a selective inhibitor of the cytokine-inducible NOS, enhanced MBP-induced EAE, but had no significant effects on myelin-induced EAN. Two other NOS inhibitors, nitro-arginine methyl-ester and N-nitro arginine, had only little or no effects in EAN and EAE. The administration of NOS inhibitors showed some striking effects in EAN and EAE, but the observed diversity of actions points to a much more complex role of the NO pathway than previously suggested.

Neuromuscular blockade by IgG antibodies from patients with Guillain-Barré syndrome: A macro-patch-clamp study

Guillain‐Barré syndrome (GBS) is often associated with serum antibodies to glycoconjugates such as GM1 and GQ1b. The pathogenic role of these antibodies and other serum factors has not yet been clarified. We have investigated the effect of serum, plasma filtrate, and highly purified IgG and IgM from 10 patients with typical GBS on motor nerve terminals in the mouse hemidiaphragm. Quantal endplate currents were recorded by means of a perfused macro‐patch‐clamp electrode. The plasma filtrate of all GBS patients led to a 5‐ to 20‐fold reduction of evoked quantal release within 7 to 15 minutes of continuous superfusion. In 4 patients, the amplitudes of single quanta were clearly reduced (by 10–66% of control values), indicating an additional postsynaptic action. Blocking effects could be reversed to a variable degree within 15 to 18 minutes after washout. Purified IgG was as effective as native serum, whereas a purified GBS IgM fraction did not block transmission. Sera from convalescent patients and IgM from healthy subjects were without blocking effect. The effects were complement independent and there was no link to the presence (in 6 patients) or absence (in 4 patients) of detectable antibodies to GM1 or GQ1b. In GBS, antibodies to an undetermined antigen depress the presynaptic transmitter release and, in some cases, the activation of postsynaptic channels. We suggest that weakness in the acute stage of GBS may be caused in part by circulating antibodies.

Circulating adhesion molecules and inflammatory mediators in demyelination: A review

Accumulating evidence shows that adhesion molecules are critically involved in inflammatory demyelination in the focusing of systemic immune responses into the target tissue, the nervous system.Adhesion molecules are upregulated through the action of cytokines. Tumor necrosis factor alpha appears to be of prime importance. Circulating adhesion molecules probably reflect acute inflammatory episodes in the central and peripheral nervous system, but may also function to modulate ongoing inflammatory responses. Cytokines released by TH 1 cells render resident and immigrant macrophages, as well as microglia, activated to synthesize and release increased amounts of inflammatory mediators, such as oxygen radicals, nitric oxide metabolites, and components of the complement system. A more detailed understanding of the sequence of immunopathologic events that culminate in myelin damage in the central and peripheral nervous systems has revealed several sites to which more specific and effective immunointervention can be targeted. NEUROLOGY 1995;45(Suppl 6): S22-S32

Different Intracellular Pathomechanisms Produce Diverse Myelin Protein Zero Neuropathies in Transgenic Mice

Missense mutations in 22 genes account for one-quarter of Charcot–Marie–Tooth (CMT) hereditary neuropathies. Myelin Protein Zero (MPZ, P0) mutations produce phenotypes ranging from adult demyelinating (CMT1B) to early onset [Déjérine-Sottas syndrome (DSS) or congenital hypomyelination] to predominantly axonal neuropathy, suggesting gain of function mechanisms. To test this directly, we produced mice in which either the MpzS63C (DSS) or MpzS63del (CMT1B) transgene was inserted randomly, so that the endogenous Mpz alleles could compensate for any loss of mutant P0 function. We show that either mutant allele produces demyelinating neuropathy that mimics the corresponding human disease. However, P0S63C creates a packing defect in the myelin sheath, whereas P0S63del does not arrive to the myelin sheath and is instead retained in the endoplasmic reticulum, where it elicits an unfolded protein response (UPR). This is the first evidence for UPR in association with neuropathy and provides a model to determine whether and how mutant proteins can provoke demyelination from outside of myelin.

Motor neuropathy in porphobilinogen deaminase–deficient mice imitates the peripheral neuropathy of human acute porphyria

Acute porphyrias are inherited disorders caused by partial deficiency of specific heme biosynthesis enzymes. Clinically, porphyrias are manifested by a neuropsychiatric syndrome that includes peripheral neuropathy. Although much is known about the porphyrias enzyme defects and their biochemical consequences, the cause of the neurological manifestations remains unresolved. We have studied porphyric neuropathy in mice with a partial deficiency of porphobilinogen deaminase (PBGD). PBGD-deficient mice (PBGD-/-) imitate acute porphyria through massive induction of hepatic delta-aminolevulinic acid synthase by drugs such as phenobarbital. Here we show that PBGD-/- mice develop impairment of motor coordination and muscle weakness. Histologically femoral nerves of PBGD-/- mice exhibit a marked decrease in large-caliber (>8 microm) axons and ultrastructural changes consistent with primary motor axon degeneration, secondary Schwann cell reactions, and axonal regeneration. These findings resemble those found in studies of affected nerves of patients with acute porphyria and thus provide strong evidence that PBGD deficiency causes degeneration of motor axons without signs of primary demyelination, thereby resolving a long-standing controversy. Interestingly, the neuropathy in PBGD-/- mice developed chronically and progressively and in the presence of normal or only slightly (twofold) increased plasma and urinary levels of the putative neurotoxic heme precursor delta-aminolevulinic acid. These data suggest that heme deficiency and consequent dysfunction of hemeproteins can cause porphyric neuropathy.

Functional abnormalities in P0‐deficient mice resemble human hereditary neuropathies linked to P0 gene mutations

Mutations in the gene encoding the transmembranous cell adhesion molecule, myelin protein zero (P0), have been reported in patients with Charcot‐Marie‐Tooth disease types 1B and 3 (Déjérine‐Sottas disease). We have previously shown that the targeted deletion of the P0 gene in mice results in impairment of sciatic nerve conduction, and we now extend our detailed electrophysiologic investigation to the facial nerve. In concordance with histologic investigations which revealed severe hypomyelination in peripheral nerves, we found the typical electrophysiologic signs of severe dysmyelination in both the facial and sciatic nerves in mice homozygously deficient for the expression of P0 (P0 −/−mice). As compared to control mice (P0 +/+), nerve conduction velocities were reduced to below 10% and compound muscle action potential (CMAP) amplitudes to below 25%, while CMAP duration and excitation thresholds were markedly increased. Surprisingly, nerve conduction changes in mice heterozygously deficient for P0 (P0 +/−) were only mild, were detected only in the sciatic nerve, and occurred not before 5–7 months of age. They were more prominent at age 12–13 months. Thus, P0 −/−mice resemble severe human inherited neuropathies like Charcot‐Marie‐Tooth disease type 3 (Déjérine‐Sottas disease) with onset early in life, whereas the P0 +/− mice may resemble the milder form, CMT1B.