Galactosphingolipids and axono-glial interaction in myelin of the central nervous system

The myelin of central and peripheral nervous system of UDP-galactose-ceramide galactosyltransferase deficient mice (cgt ^-/-) is completely depleted of its major lipid constituents, galactocerebrosides and sulfatides. The deficiency of these glycolipids affects the biophysical properties of the myelin sheath and causes the loss of the rapid saltatory conduction velocity of myelinated axons. With the onset of myelination, null mutant cgt ^-/- mice develop fatal neurological defects. CNS and PNS analysis of cgt ^-/- mice revealed (1) hypomyelination of axons of the spinal cord and optic nerves, but no apoptosis of oligodendrocytes, (2) redundant myelin in younger mice leading to vacuolated nerve fibers in cgt ^-/- mice, (3) the occurrence of multiple myelinated CNS axons, and (4) severely distorted lateral loops in CNS paranodes. The loss of saltatory conduction is not associated with a randomization of voltage-gated sodium channels in the axolemma of PNS fibers. We conclude that cerebrosides (GalC) and sulfatides (sGalC) play a major role in CNS axono-glial interaction. A close axono-glial contact is not a prerequisite for the spiraling and compaction process of myelin. Axonal sodium channels remain clustered at the nodes of Ranvier independent of the change in the physical properties of myelin membrane devoid of galactosphingolipids. Increased intracellular concentrations of free ceramides do not trigger apoptosis of oligodendrocytes.     

Beta N-acetylglucosaminyltransferase V (Mgat5) deficiency reduces the depression-like phenotype in mice

The central nervous system (CNS) is rich in glycoconjugates, located on cell surface and in extracellular matrix. The products of Golgi UDP-GlcNAc: N -acetylglucosaminyltransferases (encoded by Mgat1, Mgat2, Mgat4 and Mgat5) act sequentially to generate the GlcNAc-branched complex-type N -glycans on glycoprotein receptors. While elimination of all the branched N -glycans in Mgat1^−/− mouse embryos is lethal at neural tube fold stage, decreased branching is associated with late developmental defects similar to type 2 of congenital disorders of glycosylation, with developmental and psychomotor abnormalities. To study the role of complex-type N -glycans in brain function, we tested Mgat5^−/− mice in a battery of neurological and behavioral tests. Despite the absence of tri- and tetra-antennary products, Mgat5^−/− mice were not different from their wild-type littermates in physical and neurological assessments, anxiety level, startle reactivity and sensorimotor gating. However, they displayed a robust decrease in the immobility time in the forced swim test and the tail suspension test independent of locomotor activity, interpreted as a change in depression-like behavior. This effect was accentuated after chronic mild stress. Comparable increase in plasma corticosterone of Mgat5^+/+ and Mgat5^−/− mice in response to acute stress shows an intact function of the hypothalamus–pituitary–adrenal axis. A change in social interactions was also observed. Our results indicate that Mgat5 modification of complex-type N -glycans on CNS glycoproteins is involved in the regulation of depression-like behavior.     

A genetic screen identifies genes essential for development of myelinated axons in zebrafish

The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin.     

BRAIN CARBONIC ANHYDRASE: ACTIVITY IN ISOLATED MYELIN AND THE EFFECT OF HEXACHLOROPHENE

Abstract— Animals receiving hexachlorophene (HCP) in their diet develop cerebral edema with vacuolation of the myelin sheath. When carbonic anhydrase activities were measured in homogenates of brains from HCP-fed and control rats, the HCP-fed rats showed small decreases in the enzyme activity, but these changes were not statistically significant. HCP did inhibit the enzyme in vitro but at higher concentrations (10⁻⁵-10⁻⁴ m ) than have been reported for HCP levels in brains of experimental animals. Carbonic anhydrase activity was present in myelin preparations obtained by gradient centrifugation and osmotic shock or by subcellular fractionation. When the latter procedure was used, myelin carbonic anhydrase had a specific activity which was higher than that of the mitochondrial fraction. The myelin enzyme was inhibited by 10⁻⁹10⁻⁸ m -acetazolamide and, like the homogenates and the commercial enzyme, was inhibited by HCP. The mechanism for HCP toxicity remains unknown, but this study does suggest that carbonic anhydrase is an intrinsic component of the myelin sheath.     

Electrophysiology of mammalian Schwann cells

Schwann cells are the satellite cell of the peripheral nervous system, and they surround axons and motor nerve terminals. The review summarises evidence for the ion channels expressed by mammalian Schwann cells, their molecular nature and known or speculated functions. In addition, the recent evidence for gap junctions and cytoplasmic diffusion pathways within the myelin and the functional consequences of a lower-resistance myelin sheath are discussed.

The main types of ion channel expressed by Schwann cells are K⁺ channels, Cl⁻ channels, Na⁺ channels and Ca²⁺ channels. Each is represented by a variety of sub-types. The molecular and biophysical characteristics of the cation channels expressed by Schwann cells are closely similar or identical to those of channels expressed in peripheral axons and elsewhere. In addition, Schwann cells express P₂X ligand-gated ion channels. Possible in vivo roles for each ion channel type are discussed. Ion channel expression in culture could have a special function in driving or controlling cell proliferation and recent evidence indicates that some Ca²⁺ channel and Kir channel expression in culture is dependent upon the presence of neurones and local electrical activity.     

Behavioral characterization of mice lacking the neurite outgrowth inhibitor Nogo-A

The membrane protein Nogo-A inhibits neurite outgrowth and regeneration in the injured central nervous system, primarily because of its expression in oligodendrocytes. Hence, deletion of Nogo-A enhances regeneration following spinal cord injury. Yet, the effects of Nogo-A deletion on general behavior and cognition have not been explored. The possibility of potential novel functions of Nogo-A beyond growth inhibition is strongly suggested by the presence of subpopulations of neurons also expressing Nogo-A – not only during development but also in adulthood. We evaluated here Nogo-A ^−/− mice in a series of general basic behavioral assays as well as functional analyses related to brain regions with notable expression levels of Nogo-A. The SHIRPA protocol did not show any major basic behavioral changes in Nogo-A ^−/− mice. Anxiety-related behavior, pain sensitivity, startle reactivity, spatial learning, and associative learning also appeared indistinguishable between Nogo-A ^−/− and control Nogo-A^+/+ mice. However, motor co-ordination and balance were enhanced in Nogo-A ^−/− mice. Spontaneous locomotor activity was also elevated in Nogo-A ^−/− mice, but this was specifically observed in the dark (active) phase of the circadian cycle. Enhanced locomotor reaction to systemic amphetamine in Nogo-A ^−/− mice further pointed to an altered dopaminergic tone in these mice. The present study is the first behavioral characterization of mice lacking Nogo-A and provides significant insights into the potential behavioral relevance of Nogo-A in the modulation of dopaminergic and motor functions.     

Rubidium Uptake and Accumulation in Peripheral Myelinated Internodal Axons and Schwann Cells

Abstract: To study mechanisms of K⁺ transport in peripheral nerve, uptake of rubidium (Rb⁺), a K⁺ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero-K⁺ Ringer's solutions containing Rb⁺ (1–20 m M ) and x-ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb⁺ (Rb⁺_o) and exchanging it for internal K⁺. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1–10 m M Rb⁺_o concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb⁺ concentrations, which suggests involvement of a nonenzymatic process. At 20 m M Rb⁺_o, a differential stimulatory response was observed; i.e., axoplasmic Rb⁺ uptake velocities increased more than fivefold relative to the 10 m M rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb⁺_o uptake rate into axons and glia was completely inhibited by ouabain (2–4 m M ) exposure or incubation at 4°C. These results suggest that Rb⁺ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na⁺,K⁺-ATPase activity and implicate the presence of axonal- and glial-specific Na⁺ pump isozymes.     

Oxydative phosphorylation in sciatic nerve myelin and its impairment in a model of dysmyelinating peripheral neuropathy

Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot‐Marie‐Tooth type 1A (CMT1A) rats. The latter used as a model of dys‐demyelination. O₂ consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O₂ consumption by IMV from Wt and CMT1A 1‐month‐old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys‐demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys‐demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.     

Cytoskeletal Changes in the Brains of Mice Lacking Calcineurin Aα

Abstract: Hyperphosphorylated τ, the major component of the paired helical filaments of Alzheimer's disease, was found to accumulate in the brains of mice in which the calcineurin Aα gene was disrupted [calcineurin Aα knockout (CNAα^−/−)]. The hyperphosphorylation involved several sites on τ, especially the Ser³⁹⁶ and/or Ser⁴⁰⁴ recognized by the PHF-1 monoclonal antibody. The increase in phosphorylated τ content occurred primarily in the mossy fibers of the CNAα^−/− hippocampus, which contained the highest level of calcineurin in brains of wild-type mice. The CNAα^−/− mossy fibers also contained less neurofilament protein than normal, although the overall level of neurofilament phosphorylation was unchanged. In the electron microscope, the mossy fibers of CNAα^−/− mice exhibited abnormalities in their cytoskeleton and a lower neurofilament/microtubule ratio than those of wild-type animals. These findings indicate that hyperphosphorylated τ can accumulate in vivo as a result of reduced calcineurin activity and is accompanied by cytoskeletal changes that are likely to have functional consequences on the affected neurons. The CNAα^−/− mice were found in a separate study to have deficits in learning and memory that may result in part from the cytoskeletal changes in the hippocampus.     

On the molecular architecture of myelinated fibers

Schwann cells and oligodendrocytes make the myelin sheaths of the PNS and CNS, respectively. Their myelin sheaths are structurally similar, consisting of multiple layers of specialized cell membrane that spiral around axons, but there are several differences. (1) CNS myelin has a ”radial component” composed of a tight junction protein, claudin-11/oligodendrocyte-specific protein. (2) Schwann cells have a basal lamina and microvilli. (3) Although both CNS and PNS myelin sheaths have incisures, those in the CNS lack the structural as well as the molecular components of ”reflexive” adherens junctions and gap junctions. In spite of their structural differences, the axonal membranes of the PNS and CNS are similarly organized. The nodal axolemma contains high concentrations of voltage-dependent sodium channels that are linked to the axonal cytoskeleton by ankyrin_G. The paranodal membrane contains Caspr/paranodin, which may participate in the formation of axoglial junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated β2 subunit, as well as Caspr2, which is closely related to Caspr. The myelin sheath probably organizes these axonal membrane-related proteins via trans interactions. Accepted: 25 November 1999     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

Alteration of 5'-Nucleotidase and Na+, K+-ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (jimpy, quaking, Trembler, shiverer, and mld). Comparison with CNPase in the Developing Sciatic Nerve from Trembler

Abstract: 5'Nucleotidase and Na⁺,K⁺-ATPase are very probably myelin-associated enzymes, although not specific for this membrane. Thus, it is important to determine their activity in dysmyelinating mutants in either CNS (quaking, jimpy, shiverer, and mld) or PNS (Trembler). CNS: The activity of 5'nucleotidase was lower in mouse than in rat (10.5 and 28.0 nmol/min/mg protein in brain, respectively). In mouse myelin, the activity was 30 nmol/min/mg protein (and 72 in rat myelin). In mutants, the brain activity was very close to normal. In contrast, ATPase, the activity of which was higher in myelin as compared with forebrain homogenate, presented a reduced activity in various 21-day-old and adult mutants, except Trembler. It was normal in 8-day-old quaking and in cerebella from mutants. PNS: ATPase was lower than in brain and reduced in most mutants, this being expected for Trembler and quaking but not for shiverer and mld. 5'-Nucleotidase activity was higher compared with that in brain homogenate (relatively stable between 10-day postnatal and adult). It was affected in the mutants; in Trembler it was nearly normal in young animals but increased during development. Thus in Trembler, two different myelin-related enzymes and a myelin-specific enzyme (CNPase) presented different developmental patterns: ATPase was always reduced, 5'-nucleotidase was normal, and CNPase was slightly below normal in young (68% of the control value); CNPase activity declined during development but 5'-nucleotidase increased (42% and 190% of the control in 60-day-old animals). It is necessary to consider these results in parallel with alterations in the PNS because of Schwann cell abnormalities. Thus, determination of these two enzymes will provide a useful tool to study myelination and myelin assembly under both normal and pathological conditions.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.     

Effects of Rumpshaker Mutation on CNS Myelin Composition and Structure

Abstract: Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jp ^rsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jp ^rsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jp ^rsh myelin, further substantiating that PLP is not a component of this junctional complex. X-raydiffraction measurements on unfixed optic nerves showed that the jp ^rsh period is 5–10 Å larger than normal. Moreover, jp ^rsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jp ^rsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jp ^rsh mutant correlate with a wider periodicity and less stable packing of the myelin.     

Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice

A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium ( S.  Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S.  Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1^+/+ and Nramp1^−/− mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S.  Typhimurium to systemic sites. We observed that Nramp1^+/+ mice showed lower numbers of S.  Typhimurium in the caecum compared with Nramp1^−/− mice at all times analysed. Acute inflammation was much more pronounced in Nramp1^+/+ mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-γ, TNF-α and MIP-1α and a massive infiltration of neutrophils and macrophages, compared with Nramp1^−/− animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S.  Typhimurium infection in Nramp1^+/+ mice.     

Myelin abnormalities in mice deficient in galactocerebroside and sulfatide

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analysis to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.     

Loss of the Xeroderma Pigmentosum Group A Gene (XPA) Enhances Apoptosis of Cultured Cerebellar Neurons Induced by UV but Not by Low-K+ Medium

Abstract: To study the involvement of the xeroderma pigmentosum group A gene ( XPA ) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene ( XPA ^−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low-K⁺ medium). When cerebellar neurons from postnatal days 15–16 wild-type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m². On the other hand, neurons from XPA ^−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m². In contrast, low-K⁺ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.