Elevated specific activity of 5′-nucleotidase in a spinal cord myelin fraction from shiverer mice comparison with other myelin-associated enzymes and myelin proteins

Meylin partially purified from spinal cords of dysmyelinating mutant (shiverer) mice had almost three-fold the specific activity of 5′-nucleotidase found in the respective myelin fraction from normal mice. The specific activities of two other normally myelin-associated enzymes, 2′,3′-cyclic nucleotide-3′-phosphohydrolase and carbonic anhydrase, were only slightly higher in the myelin membranes from shiveres, compared to those from controls. In the mutants, the three enzymes probably occur in oligodendrocyte processes. Hhypothetically, the 5′-nucleotidase in the myelin sheath in shiverer and normal mice may be localized in specialized structures.     

Effect of lipid peroxidation on Na+,K(+)-ATPase, 5'-nucleotidase and CNPase in mouse brain myelin

In the presence of H2O2, solutions of Fe2+ were applied to brain homogenate and isolated myelin from adult SWV control mice and the shiverer dysmyelinating mutant mouse as a source of a reactive oxygen species (Fenton reaction). Under these conditions, lipid peroxidation was initiated and measured as thiobarbituric acid-reactive oxidation products (TBAR). This was accompanied by 85% inhibition of myelin-associated Na+,K(+)-ATPase and 25% inhibition of 5'-nucleotidase. In contrast, CNPase activity was not altered. Studies on the shiverer mutant brain revealed that in spite of hypomyelination and prevalence of premature, myelin-like membranes in the homogenate, the myelin-related enzymes reacted as normal enzymes to peroxidation. Differences in the resistance of Na+,K(+)-ATPase to peroxidation in the brain homogenate and myelin suggest that the myelin enzyme is extremely sensitive to reactive oxygen toxicity.     

Expression of a myelin basic protein gene in transgenic shiverer mice: correction of the dysmyelinating phenotype

Mice homozygous for the autosomal recessive mutation shiverer (shi) lack myelin basic protein (MBP) and exhibit a distinct behavioral pattern including tremors (shivering), convulsions, and early death. We have previously demonstrated that shiverer mice have a partial deletion in the gene encoding MBP. We now have introduced the wild-type MBP gene into the germ line of shiverer mice by microinjection into fertilized eggs. Transgenic shiverer mice homozygous for the introduced gene have MBP mRNA and protein levels that are approximately 25% of normal, and produce compacted myelin with major dense lines. Correct temporal and spatial expression of the MBP gene is achieved with a genomic MBP cosmid clone containing 4 kb of 5' flanking sequence and 1 kb of 3' flanking sequence. Moreover, the four different forms of MBP produced by alternative patterns of RNA splicing are present. These homozygous transgenic shiverer mice no longer shiver nor die prematurely.     

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.     

Biochemical Abnormalities in Spinal Cord Myelin and CNS Homogenates in Heterozygotes Affected by the Shiverer Mutation

Myelin was purified from the spinal cords of normal mice and mice heterozygous for the shiverer mutation, and measurements were made of the major myelin proteins and lipids and the specific activities of three myelin-associated enzymes. The myelin purified from the spinal cords of the heterozygotes (shi/+) was deficient by 30-40% in yield and had an apparently unique composition. In particular, when compared with normal mouse spinal cord myelin, there were more high-molecular-weight protein, less myelin basic protein, a higher protein-to-lipid ratio, and higher specific activities of 2',3'-cyclic nucleotide-3'-phosphohydrolase (EC 3.1.4.37) and carbonic anhydrase (EC 4.2.1.1) in the myelin purified from the shi/+ animals. These abnormalities were reflected in the composition of shi/+ whole spinal cord, where the protein-to-lipid ratio was intermediate between the respective values for +/+ and shi/shi spinal cords. Whole brains from shi/+ mice showed deficiencies in galactocerebroside and galactocerebroside sulfate and an increase in total phospholipid, and the lipid composition in the brains of the shi/shi mice was similar to that reported for another dysmyelinating mutant, quaking. The findings provide the first values for the lipids in normal mouse spinal cord myelin and show that heterozygotes are affected by the shiverer mutation. The observations imply that there can be considerable deviation from the normal CNS myelin content and composition without apparent qualitative morphological abnormalities or loss of function and that the amount of myelin basic protein available during myelination may influence the incorporation of other constituents into the myelin membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

Cell lineage analysis of Schwann cells in the PNS determined by shiverer-normal mouse chimeras

The myelin of the peripheral nervous system from the shiverer mutant mice is characterized by the absence of myelin basic protein, while the other myelin protein components are present at normal levels. Myelin lamella formation is normal in the shiverer mutant. Therefore, by using antiserum against myelin basic protein, we can distinguish the shiverer from the wild-type control myelin immunohistochemically. To study the cell lineage of Schwann cells, chimeras produced by the aggregation of eight-cell embryos from wild-type mice and shiverer mice have been used. Using myelin basic protein as a marker, it was observed that Schwann cells in the sciatic nerve existed as patches of cells with like-genotype. The patches occurred in a linear array along the axons with some intermingling of Schwann cells. Complete randomization by intermingling of Schwann cells was not observed and clones of Schwann cells may persist as contiguous groups throughout peripheral nerve development.     

Expression of myelin protein genes in the developing brain

The major myelin proteins fall into two classes, the basic proteins and the proteolipid proteins. In mice, five forms of the myelin basic protein (MBP) have been identified with apparent molecular masses of 21.5 kD, 18.5 kD, 17 kD and 14 kD. The 17 kD MBP variant consists of two molecular forms with similar molecular masses but different amino acid sequences. Cell-free translation studies and analyses of MBP cDNAs have shown that each of the MBP variants is encoded by a separate mRNA of approximately 2 000 bp. The five mouse MBP mRNAs appear to be derived by alternative splicing of exons 2, 5, and 6 of the MBP gene. cDNAs encoding four forms of MBP have been isolated from a human fetal spinal cord library. The mRNAs corresponding to these cDNAs are probably derived by alternative splicing of exons 2 and 5 of the human MBP gene. Proteolipid protein (PLP) cDNAs have been isolated from several species and used to establish that the size of the major PLP mRNA is approximately 3 kb. Multiple size classes of the PLP mRNAs exist in mice and rats whereas the 3 kb mRNA is the predominant form in the developing human spinal cord. In normal mice, maximal expression of the PLP gene lags behind that of the MBP gene by several days. Studies on dysmyelinating mutants have determined some of the molecular defects with respect to these two classes of myelin proteins. For example, there is a deletion of a portion of the MBP gene in the shiverer mutant. In the quaking mutant, the expression of both classes of myelin proteins is significantly reduced prior to 3 weeks. However, after 3 weeks, MBP expression approaches normal levels but the newly synthesized protein fails to be incorporated into myelin. In the jimpy mutant, although the expression of both classes of proteins is reduced, PLP expression is most severely affected.     

Effects of ZnCl2 on membrane interactions in myelin of normal and shiverer mice

X-ray diffraction was used to record the effects of metal cations on the structure of peripheral nerve myelin. Acidic saline (pH 5.0) either with or without added metal cations caused myelin to swell by 10-20 A from its native period of 178 A. The X-ray patterns usually showed broad reflections, and higher orders were either weak or unobserved. With added ZnCl2, however, the swollen myelin gave diffraction patterns that retained sharp reflections to approx. 15 A spacing. Alkaline saline (pH 9.7) containing ZnCl2 produced a reduction of the myelin period by approx. 5 A which was at least twice as much as that produced by other metals. To examine the underlying chemical basis for these unique interactions of Zn2+ with myelin, we carried out parallel X-ray experiments on sciatic nerve from the shiverer mutant mouse, which lacks the major myelin basic proteins. Shiverer myelin responded like normal myelin to ZnCl2 in acidic saline; however, in alkaline saline shiverer myelin showed broadened X-ray reflections which indicated disordering of the regularity of the membrane arrays, and additional reflections were recorded which indicated lipid phase separation. This breakdown may come about by the binding of Zn2+ to negatively-charged lipids which could be more exposed due to the absence of myelin basic proteins. Electron density profiles were calculated on the assumption that, except for changes in their packing, the myelin membranes were minimally altered in structure. For both normal and shiverer myelins, treatments under acidic conditions resulted in swelling at the extracellular apposition and a slight narrowing of the cytoplasmic space. This swelling is likely due to adsorption of protons and divalent cations. Interaction between Zn2+ and myelin P0 glycoprotein could preserve an ordered arrangement of the apposed membrane surfaces. Alkaline saline containing ZnCl2 produced compaction at the cytoplasmic apposition in both normal and shiverer myelins possibly through interactions with a portion of P0 glycoprotein which extends into the cytoplasmic space between membranes.     

Developmental dissociation of myelin synthesis and “myelin-associated” enzyme activities in the shiverer mouse

Developmental changes in three enzymes associated with myelin lipids were studied in the shiverer mouse, a murine mutant showing a severe deficiency of CNS myelin. Age-related changes in cerebroside sulfotransferase (measured in brain) and arylsulfatase A and cerebroside B-galactosidase (measured in brain and liver) were the same for shiverer and control mice. The shiverer mouse, therefore, demonstrates a dissociation between the genetic mechanisms regulating myelination in the CNS and developmental changes in enzyme activities thought to be closely related to the synthesis of myelin. In addition, we found no defect in the shiverer mouse in the incorporation of glycine-labeled basic protein into CNS myelin, indicating an important metabolic difference between the morphologically similar shiverer and quaking mutants.     

Characterization of cloned cDNA representing rat myelin basic protein: absence of expression in brain of shiverer mutant mice

A cDNA library was constructed from mRNA isolated from the brains of 18-day-old rats, the age at which myelin biosynthesis is maximal. A synthetic DNA probe synthesized based on reverse translation of the amino acid sequence of rat myelin basic protein (MBP) was used to select two cDNA clones encoding MBP. A 1.5 kb Eco RI fragment from one clone was completely sequenced. When translated, a portion of this sequence was identical at 126 of 127 positions with the reported amino acid sequence for small MBP from the rat. Brains from mice of the homozygous shiverer genotype contained neatly reduced amounts of MBP mRNA relative to wild type. A deletion of MBP sequences in the genome of shiverer mice was also demonstrated. cDNAs for MBP will allow molecular investigation of the role this gene plays in both dysmyelinating and demyelinating diseases, as well as questions of MBP biosynthesis.     

Acylgalactosylceramides in Developing Dysmyelinating Mutant Mice

Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their R_f values into two main fractions which co-nigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography-mass spectrometry analysis of both ester- and amide-linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.     

The role of myelin in Theiler's virus persistence in the central nervous system

Theiler's virus, a picornavirus, persists for life in the central nervous system of mouse and causes a demyelinating disease that is a model for multiple sclerosis. The virus infects neurons first but persists in white matter glial cells, mainly oligodendrocytes and macrophages. The mechanism, by which the virus traffics from neurons to glial cells, and the respective roles of oligodendrocytes and macrophages in persistence are poorly understood. We took advantage of our previous finding that the shiverer mouse, a mutant with a deletion in the myelin basic protein gene (Mbp), is resistant to persistent infection to examine the role of myelin in persistence. Using immune chimeras, we show that resistance is not mediated by immune responses or by an efficient recruitment of inflammatory cells into the central nervous system. With both in vivo and in vitro experiments, we show that the mutation does not impair the permissiveness of neurons, oligodendrocytes, and macrophages to the virus. We demonstrate that viral antigens are present in cytoplasmic channels of myelin during persistent infection of wild-type mice. Using the optic nerve as a model, we show that the virus traffics from the axons of retinal ganglion cells to the cytoplasmic channels of myelin, and that this traffic is impaired by the shiverer mutation. These results uncover an unsuspected axon to myelin traffic of Theiler's virus and the essential role played by the infection of myelin/oligodendrocyte in persistence.     

Endogenous cyclic AMP-stimulated phosphorylation of a Wolfgram protein component in rabbit central-nervous-system myelin.

Cyclic AMP-stimulated phosphorylation of membrane proteins in central-nervous-system myelin was investigated, with rabbit brain myelin. Subfractionation of a myelin membrane preparation by sucrose-density-gradient centrifugation produced a rapidly sedimenting population of membrane vesicles containing 5'-nucleotidase and acetylcholinesterase, a light membrane fraction containing myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase, and an intermediate membrane fraction containing the highest specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase and a small proportion of myelin basic protein. Cyclic AMP stimulation of protein phosphorylation was confined to a protein of Mr 49 700, which co-electrophoresed with the upper component of the Wolfgram protein doublet. Cyclic AMP did not affect the phosphorylation of myelin basic protein. Cyclic AMP-stimulated phosphorylation of this protein followed 2',3'-cyclic nucleotide 3'-phosphodiesterase activity on subcellular fractionation and was correspondingly high in the intermediate or 'myelin-like' fraction on sucrose-density-gradient centrifugation.     

Carbonic Anhydrase, 5''-Nucleotidase, and 2'',3''-Cyclic Nucleotide-3''-Phosphodiesterase Activities in Oligodendrocytes, Astrocytes, and Neurons Isolated from the Brains of Developing Rats

The activities of three myelin-associated enzymes, carbonic anhydrase, 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brain of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three- to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane-bound enzyme 5'-nucleotidase than was observed in neurons. In oligodendrocytes from 10- and 20-day-old rats, the 5'-nucleotidase activity was two-to threefold the activity in the homogenates (i.e., relative specific activity = 2.0-3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5'-nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10-day-old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.     

Automated discrimination of brain pathological state attending to complex structural brain network properties: the shiverer mutant mouse case

Neuroimaging classification procedures between normal and pathological subjects are sparse and highly dependent of an expert''s clinical criterion. Here, we aimed to investigate whether possible brain structural network differences in the shiverer mouse mutant, a relevant animal model of myelin related diseases, can reflect intrinsic individual brain properties that allow the automatic discrimination between the shiverer and normal subjects. Common structural networks properties between shiverer (C3Fe.SWV Mbp^shi/Mbp^shi, n = 6) and background control (C3HeB.FeJ, n = 6) mice are estimated and compared by means of three diffusion weighted MRI (DW-MRI) fiber tractography algorithms and a graph framework. Firstly, we found that brain networks of control group are significantly more clustered, modularized, efficient and optimized than those of the shiverer group, which presented significantly increased characteristic path length. These results are in line with previous structural/functional complex brain networks analysis that have revealed topologic differences and brain network randomization associated to specific states of human brain pathology. In addition, by means of network measures spatial representations and discrimination analysis, we show that it is possible to classify with high accuracy to which group each subject belongs, providing also a probability value of being a normal or shiverer subject as an individual anatomical classifier. The obtained correct predictions (e.g., around 91.6–100%) and clear spatial subdivisions between control and shiverer mice, suggest that there might exist specific network subspaces corresponding to specific brain disorders, supporting also the point of view that complex brain network analyses constitutes promising tools in the future creation of interpretable imaging biomarkers.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.