Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis.

P0, the major peripheral nervous system (PNS) myelin protein, is a member of the immunoglobulin supergene family of membrane proteins and can mediate homotypic adhesion. P0 is an essential structural component of PNS myelin; mice in which P0 expression has been eliminated by homologous recombination (P0-/-) develop a severe dysmyelinating neuropathy with predominantly uncompacted myelin. Although P0 is thought to play a role in myelin compaction by promoting adhesion between adjacent extracellular myelin wraps, as an adhesion molecule it could also have a regulatory function. Consistent with this hypothesis, Schwann cells in adult P0-/- mice display a novel molecular phenotype: PMP22 expression is down-regulated, MAG and PLP expression are up-regulated, and MBP expression is unchanged. As in quaking viable mutant mice (qk(v)), which have uncompacted myelin morphologically similar to that found in P0-/- mice, neither the qKI-6 or qKI-7 proteins are expressed in P0-/- peripheral nerve. In addition to these changes in gene expression in the P0 knockout, PLP/DM-20 accumulates in the endoplasmic reticulum of P0-/- Schwann cells, whereas MAG accumulates in redundant loops of uncompacted myelin, not at nodes of Ranvier or Schmidt-Lantermann incisures. Taken together, these results demonstrate that P0 is involved, either directly or indirectly, in the regulation of both myelin gene expression and myelin morphogenesis.

Abnormal expression and glycosylation of the large and small isoforms of myelin-associated glycoprotein in dysmyelinating quaking mutants.

The relative expression of large (L) and small (S) isoforms of the myelin-associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using antisera specific for L- and S-MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetylneuraminic acid residues linked alpha 2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oligosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants.

Quaking phenotype influences brain lipid-related mRNA levels.

Although lipids compose almost 80% of myelin, the influence of quaking on mRNAs encoding lipid biosynthetic enzymes and transport proteins has not been previously reported. Understanding the influence of quaking on myelin-specific and lipid-related mRNAs will be useful in determining the mechanism of the quaking defect. Stearoyl CoA desaturase (SCD) catalyzes a key step in the biosynthesis of oleic acid (C18:1, n-9), a major fatty acid in myelin. SCD, LDL receptor (LDLR) and apolipoprotein E (Apo E) mRNA levels are all reduced in neonatal quaking brains. In contrast to brain, quaking hepatic LDLR and Apo E mRNA levels are normal. These results indicate that lipid-related mRNAs are reduced in neonatal quaking brain, but the quaking liver is unaffected. The quaking defect influences gene expression in multiple cell types of glial lineage in the developing CNS.

Glycerophosphorylcholine phosphocholine phosphodiesterase activity in cultured oligodendrocytes, astrocytes, and central nervous tissue of dysmyelinating rodent mutants.

The levels of GPC phosphocholine phosphodiesterase, pNP phosphocholine phosphodiesterase, CNPase, and UDP galactose: ceramide galactosyltransferase activities were estimated with pure cultures of oligodendrocytes and astrocytes; mixed primary glial cells cultures; C-6 cells; and CNS tissue of the dysmyelinating md rat, the jimpy mouse, and the quaking mouse. The highest activity of GPC and pNP phosphocholine phosphodiesterases as with CNPase and C gal T was found in the pure cultured oligodendrocytes. C-6 cells had very low or undetectable activities for these two phosphodiesterases but possessed very high CNPase activity. The activity of GPC phosphocholine phosphodiesterase was significantly decreased in the CNS tissue of the md rat and the jimpy and the quaking mouse. Similar reductions were observed for the pNP phosphocholine phosphodiesterase, CNPase, and C gal T activities. The selective cellular enrichment in oligodendrocytes of the GPC phosphocholine phosphodiesterase activity and decreases of its activity in three dysmyelinating mutants in the same ratio as for CNPase and C gal T suggest that GPC phosphocholine phosphodiesterase is a myelin marker enzyme and it may reflect the quantity of myelin and oligodendrocyte present.

Developmental expression of glial fibrillary acidic protein and actin-encoding messages in quaking and control mice.

Quaking is a neurological mutation leading to pleiotropic phenotypic expression, the most prominent being disturbed myelin formation in the central nervous system (CNS) with minor abnormalities in the peripheral nervous system. Previous immunochemical measurements of glial fibrillary acidic protein (GFAP) revealed a marked increase in the protein in several areas of the CNS. To further characterize the regulation parameters of GFAP synthesis, we analyzed the levels of GFAP mRNA in 5 regions of the CNS, some with elevated levels of GFAP and some without. This was compared to the developmental expression of GFAP transcripts in the same regions in normal mice. To establish the specificity of the variations observed with this astroglial specific message, we conducted a similar investigation with actin RNA which is expressed by several cell types in the CNS. Both the actin and the GFAP message were found to be increased in the adult mutant throughout the CNS. In 2-year-old normal mice the messengers for both cytoskeleton proteins were expressed in a higher amount than in young adults.

Optic nerve tissue sections derived from myelin-deficient mutant mice as culture substrata for fibroblast adhesion and spreading.

The substrate properties were compared between normal and myelin-deficient central nervous system (CNS) tissues by an in vitro assay of cell attachment and spreading. Fibroblasts (3T3) were plated onto culture substrata consisting of optic nerve tissue sections cut from normal or two myelin-deficient mutant mice, Shiverer and Quaking. Optic nerve sections from either of the mutant animals supported more 3T3 fibroblast spreading and adhesion than sections derived from animals with normal myelin. These results demonstrate that CNS myelin influences the ability of cells to attach and spread and that it is the actual presence of myelin which is inhibitory rather than the presence of optic nerve axons or oligodendrocytes.

Developmental expression of 2',3'-cyclic-nucleotide 3'-phosphodiesterase mRNA in brains of normal and quaking mice.

A cDNA fragment of mouse 2',3'-cyclic-nucleotide 3'-phosphodiesterase was isolated and used as the probe for Northern blot analysis. The mRNA bands in normal mice became detectable at 10 days after birth, reached the maximum at the period of active myelination and then decreased gradually. At all stages of development studied, the mRNA bands in quaking mice were markedly reduced as compared with those in normal mice.

NCAM-180, the largest component of the neural cell adhesion molecule, is reduced in dysmyelinating quaking mutant mouse brain.

To explore the role of the neural cell adhesion molecule (NCAM) in myelination and remyelination, we studied the developmental expression of various molecular forms of NCAM in dysmyelinating quaking mouse brain as compared with normal mouse brain. Normal mouse brain expressed the several molecular forms differentially during development. Quaking showed a marked reduction in the expression of NCAM-180, which represents the largest component of NCAM, in comparison with normal brain. This suggests that the defective myelin compaction in the quaking mutation may be the result of a deficiency of NCAM-180.

Distribution of calcium-activated neutral proteinase activity in quaking mouse brain: a subcellular study.

The distribution of calcium-activated neutral proteinase (CANP) activity was examined in the subcellular fractions of quaking and control mouse brain. The CANP activity was determined in purified myelin, cytosol and pellet (P2, consisting of nuclei, mitochondria and microsomes) fractions using [14C]azocasein as substrate. The enzyme activity in quaking brain was 1.3-fold greater than control. Fifty-seven percent of the control brain activity was in purified myelin compared to only 7% in quaking myelin. The specific activity of the control purified myelin was 4-fold greater than homogenate while that of the quaking was two-fold greater. In contrast, 51% of the quaking brain activity was present in cytosol compared to only 18% in the control. Triton X-100 greatly increased the control brain activity (10-fold) while the quaking brain activity was increased by only 1.2-fold. The total calcium content in the quaking brain was greatly elevated (6-fold) compared to control. Approximately 30% of the brain 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was in quaking myelin while 77% of the CNPase activity in control brain was in myelin. These results suggest that in quaking brain much of the CANP is not incorporated into the myelin membrane and remains cytosolic.

Copper, manganese and zinc in the developing brain of control and quaking mice.

Copper, manganese and zinc were measured by flameless atomic absorption spectrophotometry in the developing brain of normal and quaking mice. The latter is a neurological mutant presenting early arrest of myelination. Copper concentration was increased by 200% between 10 and 20 days after birth and then leveled off in adult mice. Manganese concentration increased both in control mice and in quaking mice from 3 to 20 days by 200% and then decreased by 19% in control mice and 24% in quaking mice at adult age. Zinc increased by 93% in control and 173% in quaking mice between 10 and 20 days of age, and then progressively declined until 62 days. The mouse brain accumulates considerably all the 3 metals during early development. During the first 20 days, the augmentation is 6-fold for copper, 5-fold for manganese and 5.5-fold for zinc. In quaking, alterations are not very important.

In vitro acylation of myelin PLP and DM-20 in the quaking mouse brain.

Both proteolipid proteins (PLP) and DM-20 were found to be present by the immunoblot technique in myelin isolated from quaking mouse brain; however, the relative concentration of these proteins in myelin from quaking brain was substantially reduced when compared to the control. Brain slices from littermate control and quaking mice were incubated with [3H]palmitic acid to determine the incorporation of fatty acid into myelin proteolipid proteins. Fluorography of gels containing myelin proteins from control and quaking mice brain revealed that both PLP and DM-20 were acylated. The incorporation of [3H]palmitic acid into quaking myelin PLP and DM-20 was reduced by 75% and 20% respectively of those in control brain. The significance of differential acylation of quaking myelin PLP and DM-20 is discussed with respect to availability of non-acylated pools of proteolipid proteins and the activities of acylating enzymes.

Myelin-associated glycoprotein in the central and peripheral nervous system of quaking mice.

The myelin-associated glycoprotein (MAG) was quantitated in the CNS and PNS of quaking mice and the levels compared to the levels of myelin basic protein (MBP) and 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. In the brainstems of 36-day-old quaking mice, MBP, MAG, and CNPase were reduced to 12, 16, and 29% of control levels, respectively. In the sciatic nerves of the 36-day-old quaking mice, MBP and CNPase were 38 and 75% of control levels, respectively, whereas the concentration of MAG was unchanged or slightly increased. Similar quantitative results were obtained for the sciatic nerves and spinal roots of 7-month-old quaking mice. Immunoblots showed that the principal MAG band from the brainstems, sciatic nerves, and spinal roots of the quaking mice had a higher than normal apparent Mr. In addition, there was a minor component reacting with anti-MAG antiserum in the brainstems of the quaking mice that had a slightly lower Mr than control MAG and was not detected in the normal mice. The results for the quaking mice are compared with those from similar studies on other mutants with dysmyelination of the CNS and PNS.

Increased turnover of myelin proteolipid protein in quaking mouse brain.

Homozygous quaking and normal control littermate mice were injected intracerebrally with [3H]leucine at 19 days of age. The animals were sacrificed after 1 h and after 6 days. The proteolipid protein (PLP) and intermediate protein (IP) were extracted from whole brain by chloroform-methanol (2:1) and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). One hour postinjection the labeling of total protein in quaking brain was the same as the control and the radioactivity of PLP and IP in quaking was approximately 35% of the control. Six days after precursor administration the radioactivity of the total protein decreased significantly in both groups and to the same extent. However, the labeling of PLP and IP more than doubled in the control, while it decreased by half in the quaking brain. The results indicate that there is an increased turnover rate of PLP and IP in quaking brain.

Proteolysis in quaking mouse brain and spinal cord.

Six proteolytic enzymes were assayed for activity in quaking CNS in examining the hypothesis that increased proteolytic activity contributes to the hypomyelination characteristics of this mutant. Cathepsin B-like enzyme, cathepsin D, neutral proteinase, calcium-activated neutral proteinase, prolyl endopeptidase, and diaminopeptidase II were assayed in whole homogenate of brain or spinal cord and each was found to have activity similar to that in normal mice. These results do not support a relationship between proteolysis and the genetic defect and suggest that other factors should be investigated to delineate the pathogenesis of this mutant.

Altered brain copper and zinc content in quaking mice.

Brain copper and zinc levels were determined in 21-day-old and "adult" C3HeB/FeJ quaking mice and in normal littermate controls. Expressed per gram dry weight of brain, copper was increased 84% over normal mice at 21 days after birth, but was not significantly different from normal in the adults. Zinc was increased 23 to 24% at both ages. At both ages, brains from quaking mice had a significantly reduced content of solids, indicating increased water content in the mutant brain. Our study is the first to report copper and zinc content as a measure of both wet and dry brain weights. Our results indicate abnormal copper content in the quaking mutant. The relationship between copper content and other aspects of the quaking phenotype, including its seizure behavior and myelin deficit, remain to be established.

[Free and sulfo-conjugated dehydroepiandrosterone in the brain of mice with myelin biosynthesis disorders]. / Déhydroépiandrostérone libre et sulfo-conjuguée dans le cerveau de souris dysmyéliniques.

Dehydroepiandrosterone, either unconjugated (D) or conjugated to sulfuric acid (DS), has been identified in the brain of male Mice; DS had been previously found in Rat brain. DS amounts in posterior brain, were 0.8-2.0 ng/g in controls, and very much lower in dysmyelinic jimpy and quaking Mice of the same age. Conversely, amounts of D were increased in affected Mice, suggesting an impaired sulfoconjugation. Results may be explained by accumulation of D and DS in brain, unrelated to the endocrine system.

[Mutations affecting myelination in the mouse; their correlation to human diseases (author's transl)]. / Dysmyélinisations génétiques de la souris. Leur apport à la pathologie humaine.

Murine species appear to be particularly useful for studying myelinogenesis since the cell differentiation process occurs postnatally. The discovery of mutations in the mouse, whose phenotypic expression mainly involves myelination, has provided a new tool for approaching the neurobiology of myelin and its pathology.