Myelin-deficient Mutant Mice Research Articles

Effects of carbonic anhydrase II (CAII) deficiency on CNS structure and function in the myelin‐deficient CAII‐deficient double mutant mouse

In the choroid plexus carbonic anhydrase II (CAII) supports the transport of bicarbonate ions, sodium ions, and water from blood to the CSF, and in the myelin sheath CAII supports compaction of myelin by stimulating cotransport of ions and water out from between the myelin membranes. In view of the latter, it is surprising that mutant mice deficient in CAII (Car-2n) have compact myelin. Since myelin basic protein also takes part in myelin compaction, we bred double CAII-deficient, myelin-deficient (Mld) mutant mice, in which the adults would have some compact myelin sheaths and a partial deficiency in myelin basic protein, with a view to examining oligodendrocytes and myelin sheaths in the double mutant. Like the parent Mld strain, the double mutants displayed tremors and seizures; however, the onset of seizures was delayed significantly in the double mutants, and the lifespan increased by several months. Like the brains of Car-2n mutants, those of double mutants (MldCar-2n) were deficient in mRNA and protein for CAII and showed upregulation of a different isozyme, CAIV. In the double mutants, oligodendrocytes were reduced in number, and the myelin sheaths and oligodendrocytes were swollen. The partial protection against seizures, which CAII deficiency conferred, suggests that acidosis in the central nervous system (CNS) of the Car-2n and MldCar-2n mice, due to absence of CAII from the choroid plexus, may downregulate the activity of NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the axonal microtubule cytoskeleton by myelinating Schwann cells

The Trembler PNS myelin-deficient mutant mouse offers a unique model for the study of axon-glial interactions. Previous work in our laboratory on Trembler mouse sciatic nerve established that myelinating Schwann cells exert a profound effect on the underlying neuronal cytoskeleton. Demyelinated axon segments exhibited decreases in the rate of slow axonal transport, axonal caliber, and neurofilament phosphorylation, as well as increases in neurofilament density. The present study considers effects on the microtubule cytoskeleton. At least two aspects of the microtubule cytoskeleton in Trembler PNS axons were altered by demyelination. First, the stability of the Trembler axonal microtubule cytoskeleton is decreased, as measured by decreased levels of insoluble tubulin (Sahenk and Brady, 1987). Second, the composition and phosphorylation of axonal microtubule-associated proteins, including tau, MAP 1A, and MAP 1B, are changed in Trembler demyelinated nerves. Further, the fraction of axonal tubulin moving at slow component b rates was increased (de Waegh and Brady 1990, 1991). These results provide further evidence that cell-cell interactions between myelinating glia and their underlying axons extend beyond a structural role, actively influencing biochemical and physiological properties of the axon.

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.

Altered slow axonal transport and regeneration in a myelin-deficient mutant mouse: the trembler as an in vivo model for Schwann cell-axon interactions

The thickness of the myelin sheath in normal myelinated nerve is proportional to the diameter of the axon. In the demyelinating mutant mouse, Trembler, not only is the thickness of the myelin sheath reduced, but the caliber of associated axons is smaller. This correlation suggests that the interaction between axons and Schwann cells may affect the shape and function of axons as well as properties of myelin. Since axonal diameter depends in part on the cytoskeleton and its movement with slow axonal transport, we have compared the properties of slow transport in the sciatic nerve of control and Trembler mice. Studies of the sciatic nerve of normal mice showed that the rates for proteins moving in slow component a (SCa) and slow component b (SCb) are similar to those previously measured in rat. In Trembler mice, tubulin was transported significantly faster than in control mice, with a rate of 1.73 mm/d for Trembler compared to 1.56 mm/d in the control. In contrast, the rate for neurofilament proteins was significantly slower in the Trembler (1.15 mm/d compared to 1.38 mm/d in the control). The majority of proteins in SCb were also transported slower in Trembler than control: actin and calmodulin were transported at 2.29 mm/d as compared to 2.73 mm/d in control, while spectrin and clathrin were transported at 2.01 and 2.43 mm/d, respectively, as compared to 2.54 mm/d in control. The importance of slow axonal transport in regeneration has been suggested by the clear correlation between the rates of regeneration and the rates of SCb. Therefore, we evaluated regeneration of motor axons in Trembler mice to determine whether the regenerative response was affected by deficient Schwann cells. A slower regeneration rate was found in the Trembler (1.7 mm/d) motor axon when compared to the control (2.29 mm/d), but elongation of fibers in regeneration began after a shorter delay in the Trembler (1.6 d) than in control (2.5 d). Thus, deficient Schwann cells and poor myelination appear to affect both quantitative and qualitative properties of slow axonal transport. These changes lead to alterations in the morphological and physiological properties of affected axons.

Myelin/Oligodendrocyte Glycoprotein Expression during Development in Normal and Myelin-Deficient Mice

The myelin/oligodendrocyte glycoprotein (MOG) is identified by monoclonal antibody 8-18C5. MOG is localized on the surface of myelin and oligodendrocyte processes. Recently, several studies have shown that MOG plays an important role as a target for antibody-induced demyelination. In the present study, we investigated MOG expression in the brains of normal and myelin-deficient (mld) mutant mice during development. By gel electrophoresis and immunoblotting, we observed the developmental pattern of two closely migrating bands, with apparent molecular masses of 26 and 28 kilodaltons. Their concentrations increased coordinately during the most active phase of myelin and myelin basic protein (MBP) synthesis. Between 20 and 25 days of age, the MOG developmental pattern superimposed that of MBP as well as myelin yields. In mld mutant mice, which are affected by a severe deficit of MBP synthesis, MOG was present at reduced levels (40% of controls at 60 days of age). At 85 days of age, mld mice exhibited increased concentrations of MBP, and myelin was better compacted. At this age, MOG concentrations increased and reached 70% of controls. These results suggest that MOG could play a role in the maintenance or completion of the myelin sheath. Its expression level may be modulated by the presence of compact myelin and/or MBP in the myelin sheath.

The duplicated myelin basic protein gene in mld mutant mice does not impair transcription

Myelin basic protein (MBP) gene organization and expression were analyzed in wild type and myelin deficient ( mld) mutant mice. Southern analysis demonstrated MBP gene duplication in mld mice. In addition, we present evidence that one MBP gene in mld mice is normal for at least 14 kilobases (kb) upstream from exon I, whereas the second gene is normal for at least 3.5 kb but not more than 7 kb upstream from exon I. Run-on experiments showed that the rate of MBP gene transcription in mld mice is similar to that seen in normal mice. Detailed analysis of the transcriptional activity of various regions of the gene led us to conclude that all portions of the MBP gene are transcribed in mld mice. Consequently, we propose that the low levels of MBP mRNA observed in these mice (2–5% of the wild-type level) are not due to deficient transcriptional activity.

The role of myelination in learning performance observed in two strains of myelin-deficient mutant mice (shiverer and mld)

In order to study whether myelination is involved in learning performance, the behavior of myelin-deficient mutant mice (shiverer and mld) was examined. Shiverer is a deletion mutant of the myelin basic protein (MBP) gene causing severe myelin deficits due to the complete absence of MBP, while mld, allelic mutant to shiverer, shows lowered MBP expression, resulting in less severe effects. Shiverer clearly showed deficits in successive reversal learning, while mld showed less deficits in the learning performance. Both mutant mice showed no deficits in the radial maze performance, which is though to show the natural foraging behavior of rodents. These results suggest that myelin formation is related to learning but not to natural behavior.

Immunohistochemical, biochemical and electron microscopic analysis of myelin formation in the central nervous system of myelin deficient (mld) mutant mice

Myelin deficiency (mld) is an autosomal recessive mutation in mice and is considered to be allelic to the shiverer (shi) mutation. Mld mice are characterized by hypomyelination of the central nervous system (CNS). They show typical symptoms such as tremor, tonic convulsion and ataxic movement. Subcellular fractionation of the CNS revealed that the MBP bands were greatly decreased in the P2A (myelin) fraction and the total content of myelin basic protein (MBP) was much lower than that in the control in all parts of the CNS. Sections from mld mice were examined by immunohistochemical tests with MBP antiserum, and a mosaic expression of MBP was found in the myelin of the mld mice. Since the major dense line is considered to be composed mainly of MBP, we investigated the myelin of mld mice by electron microscopy and found that there were 3 types of myelin: (1) a normal type compact myelin with a major dense line, (2) a shiverer-type myelin with no major dense line, and (3) a mixed-type myelin, in which within a myelin lamella the major dense line abruptly changes to cytoplasm of oligodendrocytes.

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice.

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.

Biochemical and physicochemical determinations in a premyelin fraction obtained by zonal centrifugation in normal mouse and in dysmyelinating mutants (quaking, shiverer, and myelin-deficient).

Myelin and premyelin material denser than myelin were obtained from quaking (Qk), shiverer (Shi), and myelin-deficient (mld) mutant and control mice, using zonal centrifugation on zonal rotor. On these fractions, we performed biochemical analysis (lipids and fatty acid), and, in parallel, we determined the physical structure of membranes by the spin-label method. The hyperfine splitting constant (2 Tll) was used to determine the order of membranes and their rigidity, and frequency of rotation (Vc) was used to measure fluidity. In control mice, the premyelin material contained a lesser amount of sphingolipids than pure myelin, but the relative proportions between hydroxy- and nonhydroxy-cerebroside and sulfatides were similar in the premyelin material and in pure myelin. The premyelin material contained half the alkanes found in the pure myelin and much less very-long-chain fatty acids. The (2 Tll) was lower in the premyelin material, but the (Vc) was similar in myelin and premyelin material. In mutants, the amount of material recovered in the premyelin fraction was reduced in qk, and increased in both shi and mld. The relative amount of sphingolipids were normal in mld, but not in shi mutants, especially in cerebrosides formed with alpha-hydroxylated fatty acids and sulfatides formed with unsubstituted fatty acids. The absolute amounts of sphingolipids were nearly normal in both shi and mld. In the premyelin fraction from qk mutants, both relative and absolute amounts of sphingolipids were drastically altered. In percentage, cerebrosides and sulfatides formed with nonhydroxyfatty acids were dramatically reduced, and, conversely, cerebrosides and sulfatides formed with hydroxyfatty acids were increased. In terms of absolute amount, only cerebrosides and sulfatides formed with nonhydroxyfatty acids were dramatically reduced. In the premyelin fraction, polyunsaturated fatty acids were increased in shi and mld, but decreased in qk. In this mutant, lignoceric (24:0) and nervonic (24:1) acids were drastically reduced. The amount of alkanes in the premylin material from qk and mld was reduced by 50%. The shi fraction was nearly free of alkanes. The maximal apparent coupling constant (hyperfine splitting constant, 2 Tll) was not affected in the mld and qk mutant, but was reduced in the shi mutant premyelin fraction. The Vc was dramatically increased in the qk, slightly decreased in the shi, and close to control in the mld. This work provides additional data on premyelin material prepared in various neurological mutants using continuous gradients in zonal rotor.(ABSTRACT TRUNCATED AT 400 WORDS)

Dysmyelination in <I>Shiverer</I> and <I>Myelin Deficient</I> (<I>mld</I>) Mutant Mice

Dysmyelination in <I>Shiverer</I> and <I>Myelin Deficient</I> (<I>mld</I>) Mutant Mice

Anticonvulsive treatment of myelin-deficient (mld) mice improves survival and confirms the delayed increase of myelin basic protein.

Myelin-deficient (mld) mutant mice were treated with phenobarbital between 60 and 90 d of age. The survival rate at 90 d increased from 1.4% in untreated mutants to 46% in those who received phenobarbital. This is evidence that apneic spells during tonic seizures are a major cause of death in mld mice. Myelin basic protein (MBP) content of brain homogenates from treated mld mice increased significantly between 30 and 90 d. MBP was present in myelin purified from the 90-d-old treated mld mice. These results demonstrate that the MBP increase, which occurs after the active phase of myelin formation is completed, is a general phenomenon and is not caused by the selection of a small and mildly affected subpopulation of mutants.

Myelination in the CNS of mld mutant mice: Comparison between composition and structure

Myelination was studied between 15 and 135 days postnatally in the brain and optic nerves of myelin deficient ( mld) mutant mice. Between 15 and 30 days almost no myelin basic protein (MBP) could be detected in mld myelin. The axons were loosely wrapped by membranes which only fused at the extracellular sites forming the intraperiod line. At this age the major dense line was absent. At 25–30 days, purified myelin contained extremely high 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNP) (EC 3.1.4.37) activities which could be related to the redundant paranodal-like structures observed at this age in mld CNS. Therefore, it can be suggested that CNP is probably localized in such paranodal loops. After the active phase of myelin deposition was completed in controls, mld mutants showed important increases of MBP concentration in myelin with the concomitant appearance of the major electron dense line and better compaction of the myelin lamellae. The yield of myelin increased from 5 to 14% of control values during the period of 30 to 135 days. Since the recovery phase occurred at the time when myelin lipid synthesizing enzymes are at low residual activities, the myelin deficit could only be partially corrected. This study indicates that there is a delay of MBP synthesis in mld mice and the decrease of other myelin proteins could be secondary to the assumed primary defect involving MBP.

Axonal swellings in jimpy mice: Does lack of myelin cause neuronal abnormalities?

We have observed focal axonal enlargements in jimpy, a myelin deficient mutant mouse. Similar axonal swellings have also been found in other studies, in two other myelin deficient mutant mice and in a myelin deficient mutant rat. We suggest that this axonal abnormality represents a common secondary reaction to lack of myelin. Such a secondary reaction might also occur in other species including human, in response to deficient myelin or to loss of myelin due to disease.

In vitro myelin basic protein synthesis in the PNS and CNS of myelin deficient ( mld) mutant mice

The CNS of the mutant mld has a severe myelin deficit. In contrast, myelination is normal in the PNS. Previous studies showed that myelin basic proteins (MBP) are practically missing in mld CNS and PNS tissues. Using an in vitro system and immunoprecipitation, we present evidence that MBP synthesis is repressed in the PNS and in CNS of mld mutants.

Intrinsic myelin proteins are normally synthesized in vitro in the myelin-deficient (mld) mutant mouse.

The synthesis of myelin basic protein is severely reduced in the central and peripheral nervous system of myelin-deficient (mld) mutant mice. Using an in vitro system and immunoprecipitation, we found a normal rate of synthesis in mld mice for two intrinsic myelin proteins: proteolipid protein in central nervous system myelin and P0 in peripheral nervous system myelin. These results indicate that protein synthesis on membrane-bound ribosomes is not affected by the mutation.

Modifications of ganglioside composition in peripheral nerve of myelin deficient Trembler mutant mouse.

Ganglioside distribution was studied in peripheral nerves of normal controls and those of Trembler mutant mouse with defect in Schwann cell differentiation and myelination. Neuraminic acid content was considerably decreased in the mutant. Ganglioside distribution as evaluated by densitometry of resorcinol positive spots on thin-layer chromatography revealed a major peak for GD1a in normal controls. In the mutant, the relative proportion was modified with qualitative modifications in the GD1a area and a tremendous increase in GM3 content. The relation with the intense Schwann cell proliferation observed in the mutant is discussed.

Shiverer peripheral myelin contains P2.

Myelin-deficient mutant mice, such as shiverer, can provide information about the normal mechanisms involved in myelination. The shiverer mouse carries a recessive, autosomal mutation resulting in an extreme deficiency in central myelin, and the small amount of myelin present is poorly compacted; the peripheral myelin, however, appears essentially normal. As the amount of myelin basic protein (P1) in both central and peripheral nervous system myelin is extremely low in shiverer, it is possible that P1 is essential for the normal formation and compaction of central myelin, but not of peripheral myelin. Some other protein would then be responsible for the formation of compact peripheral myelin in shiverer. Peripheral myelin contains another basic protein, designated P2, which could be a possible candidate for this role. Kirschner and Ganser, however, using SDS-polyacrylamide gel electrophoresis, reported that P2, as well as P1, is absent from shiverer sciatic nerve. This is an important observation if correct, because it not only excludes the possibility that P2 is required for compaction but also makes it less likely that the deficiency in P1 is the primary defect in shiverer. As P2 in rat and mouse has frequently been confused with another small basic protein (related to P1) in SDS-polyacrylamide gels, it seemed worthwhile to reassess this aspect of the Kirschner and Ganser observations. Immunohistochemistry and immunoblotting have been used here to show unambiguously that P2 is present in shiverer peripheral myelin.

Increased axonal proteolysis in myelin-deficient mutant mice.

Protein degradation within retinal ganglion cell axons in vitro is 50 to 110 percent faster than normal in mutant mice exhibiting deficiencies of myelin in the central nervous system. Proteolysis is increased proximally and distally within retinal ganglion cell axons of mice carrying the jumpy mutation or its allele, myelin synthesis deficiency, and is increased distally within those axons of quaking mice. The proteolytic defect is axon (neuron)-specific since the rate of protein degradation within glial cells is normal. Increased axonal proteolysis does not bear a simple relation to hypomyelination since shiverer, another mouse mutant deficient in central myelin, displayed normal rates of axonal protein degradation under the same conditions. These observations suggest an abnormal axon-glial interaction in mice with primary glial defects and raise the possibility that the functioning of histologically normal axons (neurons) may be altered in dysmyelinating diseases.

Similarities and dissimilarities between two myelin deficient mutant mice, Shiverer and mld

In the brain of Shiverer and mld mutant mice, myelin is poorly compacted and the major dense line of the myelin is practically missing. Major biochemical differences were detected between mutations. In mld myelin, myelin basic proteins are mainly affected andd 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNP) exhibits a very high specific activity. In Shiverer myelin, in addition to basic proteins, all major myelin proteins are also decreased while CNP specific activity is moderately increased.