Myelin Deficient Mutant Research Articles

High b-value q-space diffusion MRI in myelin-deficient rat spinal cords

In this study, we explore the effect of the lack of myelin on the diffusion characteristics and diffusion anisotropy obtained from high b-value q-space diffusion-weighted MRI (q-space DWI) in excised rat spinal cords. Twenty-one-day-old myelin-deficient (md) mutant (N=6) and control rats (N=6) were used in this study. The MRI protocol included multi-slice T(1), T(2), proton density (PD) MR images and high b-value q-space diffusion MRI measured perpendicular and parallel to the fibers of the spine. q-Space displacement and probability maps, in both directions, as well as displacement anisotropy maps, were computed from the diffusion data. At the end of the MRI protocol, representative spinal cords from both groups were subjected to electron microscopy (EM). The md spinal cords show different gray/white matter contrast in the T(1), T(2) and PD MR images as compared with controls. In addition, the mean displacement extracted from the high b-value q-space diffusion data was found to be dramatically higher in the white matter (WM) of the md spinal cords than the controls when diffusion was measured perpendicular and parallel to the fibers of the spine. However, interestingly, at the diffusion time used in the present study, the difference in the WM displacement anisotropies of the two groups was not found to be statistically significant. Myelin was found to have a pronounced effect on the diffusion characteristics of water in WM but less so on the diffusion anisotropy observed at the diffusion time used in the present study.

Role of myelination in the development of a uniform olivocerebellar conduction time.

Purkinje cells generate simultaneous complex spikes as a result of olivocerebellar activity. This synchronization (to within 1 ms) is thought to result from electrotonic coupling of inferior olivary neurons. However, the distance from the inferior olive (IO) varies across the cerebellar cortex. Thus signals generated simultaneously at the IO should arrive asynchronously across the cerebellar cortex, unless the length differences are compensated for. Previously, it was shown that the conduction time from the IO to the cerebellar cortex remains nearly constant at approximately 4 ms in the rat, implying the existence of such compensatory mechanisms. Here, we examined the role of myelination in generating a constant olivocerebellar conduction time by investigating the latency of complex spikes evoked by IO stimulation during development in normal rats and myelin-deficient mutants. In normal rats, myelination not only reduced overall olivocerebellar conduction time, but also disproportionately reduced the conduction time to vermal lobules, which had the longest response latencies prior to myelination. The net result was a nearly uniform conduction time. In contrast, in myelin-deficient rats, conduction time differences to different parts of the cerebellum remained during the same developmental period. Thus myelination is the primary factor in generating a uniform olivocerebellar conduction time. To test the importance of a uniform conduction time for generating synchronous complex spike activity, multiple electrode recordings were obtained from normal and myelin-deficient rats. Average synchrony levels were higher in normal rats than mutants. Thus the uniform conduction time achieved through myelination of olivocerebellar fibers appears to be essential for the normal expression of complex spike synchrony.

Oligodendrocyte survival and function in the long-lived strain of the myelin deficient rat

This study has examined cellular and molecular aspects of glial cell function in a newly described long-lived myelin deficient rat mutant. In contrast to the shorter-lived mutants which died at 25-30 days, the longer-lived mutant rats lived to 75-80 days of age. Despite living longer, these mutants had a similar frequency of seizures to their younger counterparts. In the spinal cord and optic nerves of the older mutants, myelinated fibres in similar numbers to those seen in the younger myelin deficient rats were present. However, the total glial cell numbers were markedly reduced with few remaining normal appearing oligodendrocytes, and very few microglia compared to the younger mutants. In addition, little or no cell death or division was seen in the longer-lived rats. However, there was some evidence of ongoing myelination and the persistence of immature oligodendrocytes or their progenitors in the older mutant. There was some continued myelin gene expression, although this was at much reduced levels compared to normal, with proteolipid protein and myelin basic protein being most affected. In situ hybridization analysis for proteolipid protein mRNA showed that few proteolipid protein expressing oligodendrocytes remained in the 70-80-day-old mutant. Polymerase chain reaction analysis of exon 3 of the long-lived mutant revealed the same point mutation as described in the younger myelin deficient rat.

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.

Ontogeny of glycerol phosphate dehydrogenasepositive oligodendrocytes in rat brain. Impaired differentiation of oligodendrocytes in the myelin deficient mutant rat

The ontogeny of oligodendrocytes in the myelin deficient (md) rat mutant and in control rats was explored immunohistochemically using an antiserum against the oligodendrocyte specific enzyme, glycerol phosphate dehydrogenase (GPDH), and the avidin-biotin complex technique. In control rats, GPDH was demonstrated to be expressed relatively early in oligodendrocyte differentiation, prior to either myelin basic protein or proteolipid protein expression. With development, oligodendrocytes containing GPDH increased in number, apparent staining intensity, cell soma area and process elaboration. Fewer GPDH+oligodendrocytes were observed in the brain of mutant rats than in unaffected littermates at all developmental ages, and major developmental increases in oligodendrocyte density were delayed. The density of GPDH+oligodendrocytes was reduced by about 40% in both the corpus callosum and in the cingulate cortex of P22–25 md mutants compared with control rats. The oligodendrocyte cell soma area was not influenced by the md condition, and increased 2-fold with development in rats of both genotypes. The area of coronal sections occupied by the corpus callosum increased about 2.5-fold with development, and was 30% smaller in mutant rats late in their lifespan than in unaffected littermates. The reductions in oligodendrocyte density reported here are of insufficient magnitude to fully account for biochemically measured reductions in oligodendrocyte gene expression accompanying the md trait, indicating that gene expression per oligodendrocyte is also impaired. Cell counts in control rats also revealed that oligodendrocytes are overproduced during development. Cell density and the total number of corpus callosum GPDH+oligodendrocytes per section were maximal at P22–25 and then decreased to adult values. These results suggest that glial cells, like neurons, may be generated in excessive numbers, and some subsequently die, as a normal concomitant of development.

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.

Molecular biology of myelin basic protein: Gene rearrangement and expression of anti-sense RNA in myelin-deficient mutants

1. Myelin is an important structure for facilitating the conduction of impulses along the axons both in the central nervous system (CNS) and peripheral nervous system (PNS). 2. Myelin basic protein (MBP) is a major protein in CNS myelin. 3. MBP is expressed specifically in the nervous system. 4. The MBP gene has been cloned and characterized. 5. Two mutant mice, Shiverer ( shi) and myelin-deficient ( mld, shi mid ), are autosomal recessive mutants that show severe symptoms such as intentional tremor. They have been found to have a mutation in the MBP gene that results in poor myelination in the central nervous system. 6. It was found that rearrangement within the MBP gene results in low expression of the gene. 7. In Shiverer, the MBP gene is partially deleted (from exons 3 to 7), and in mld, the gene is duplicated tandemly and a large portion of the duplication is inverted upstream of the intact copy. 8. In mld, anti-sense RNA complementary to exons 3–7, which correspond to the inverted segment, was detected by RNase protection studies, and presumed to be responsible for the reduced expressions of MBP. 9.The mechanism of gene rearrangement in MBP was also characterized. 10. This article reviews the recent progress in the study of the MBP gene, especially the rearrangement of the gene and its expression in mutant mice.

ADP-ribosylation of myelin basic proteins isolated from normal and mutant mouse brains

Myelin basic proteins (MBPs) have been shown to be ADP-ribosylated in-vitro by cholera toxin in the presence of NAD. Since acid-soluble extracts of brain contain other proteins in the 14-32 kD range (such as histones) in addition to MBP's, the identification of the ADP-ribosylated proteins was uncertain. To determine that only the MBP's were ADP-ribosylated, the acid-soluble fractions from several murine mutants were prepared. Thus, in the Shiverer mutants, none of the proteins in the 14-32 kD range were ADP-ribosylated; in the Myelin-deficient mutant, some of ADP-ribosylation was observed but none in the Jimpy mutant, consistent with our demonstration that the least cationic isomer cannot be ADP-ribosylated.

Chimeric and Molecular Genetic Analysis of Myelin‐Deficient (Shiverer and Mld) Mutant Mice

The shiverer and myelin-deficient (mld) mutants are two allelic mutations. Both are characterized by hypomyelination in the CNS and deficient expression of the MBP gene. Chimeric analysis of the pathogenesis of shiverer showed that shiverer mutation acts intrinsic to the oligodendrocyte, which is the only cell type expressing the MBP gene in the CNS. Molecular genetic studies by several groups demonstrated that shiverer is a deletion mutation in the MBP gene. Consequently, no MBP is produced in the shiverer mutant. In mld, however, partial expression of the MBP gene was observed. Interestingly, MBP was expressed in a mosaic fashion in the CNS of mld mice. Molecular genetic studies revealed that the mld mutant has duplicated MBP genes in tandem on a distal part (E2-4) of chromosome 18q. The reduced expression was based on the level of mRNA. A large portion is inverted in the upstream copy, and an intact copy is located downstream. We showed in mld mutants that antisense RNA corresponding to the inverted segment is transcribed, and it forms RNA duplex, with the RNA transcribed from the normal gene located downstream.

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.

Extracellular potassium activity and axonal conduction in spinal cord of the myelin-deficient mutant rat

We recorded somatosensory evoked potentials (SEPs), extracellular K + ionic activity ([K +] e), and K + clearance rates in the spinal cords of 14 myelin-deficient mutant rats and 16 normal male littermates at 16–41 days after birth. Tested under pentobarbital anesthesia (25 mg/kg ip) and hypothermic conditions (32–34°C), myelin-deficient rats had longer cortical SEP latencies (67 ± 20 ms) compared to those in normal siblings (48 ± 15 ms; P < 0.05). Mean baseline [K +] e levels were 2.6 ± 0.5 m M in myelin-deficient rats and 2.6 ± 0.8 m M in normal siblings. Clearance times of KCl solutions injected into the spinal cord were biphasic and exponential. The mean initial and secondary exponential half-times were 1.0 ± 0.5 and 2.7 ± 1.7 min for myelin-deficient rats and 0.8 ± 0.4 and 3.8 ± 3.2 min for normal siblings. Repetitive sciatic nerve stimulation (2–20 Hz, 2- to 6-s trains) produced 1–3 m M transient [K +] e rises in thoracic and lumbar cords of myelin-deficient rats. The [K +] e rises were largest in the dorsal spinal cord at 200–500 μm depth. The normal siblings had smaller or no stimulus-induced [K +] e rises. In myelindeficient rats, injection of 1 m M 4-aminopyridine (4-AP) solution into the thoracic spinal cord completely suppressed the stimulus-induced [K +] e and markedly increased spinal and cortical SEP amplitudes for several hours. In the normal siblings, the 4-AP injections transiently blocked spinal conduction for 20–30 min but thereafter enhanced cortical SEP amplitudes for 2–3 h. We conclude that sciatic nerve stimulation produces spinal cord [K +] e rises in myelin-deficient rat larger than those in the normal siblings, that the [K +] e transients represent increased K + release rather than impaired K + clearance, and that the K + ions come from 4-AP blockable sources.

Spinal cord lesions block seizures or delay their onset in myelin-deficient rats: Evidence that generalized tonic seizures can be triggered by abnormal spinal cord activity

The myelin-deficient mutant rat develops generalized tonic seizures and dies during the fourth postnatal week. Surgical constriction of the lower thoracic spinal cord, performed either after the seizures have appeared or before, eliminates the seizures, or delays their onset, and prolongs the life of the animals. These observations support the view that the seizures in these animals can be triggered by abnormal activity originating in the myelin-deficient spinal cord and can be blocked by preventing that activity from ascending to higher levels. Similar seizures and “paroxysmal” phenomena occur in other myelin-deficient conditions including multiple sclerosis.

Immunocytochemical Staining of Astrocytes, Tanycytes, Immature Glial Cells and Ependymal Cells in the Central Nervous System of the Normal Rat and the Myelin-Deficient Mutant

Central nervous system (CNS) tissue from young myelin-deficient (md) mutant rats and male littermate controls was immunostained with antibodies against the following glial-cell antigens: glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin, the Yb isozyme of glutathione-S-transferase (Yb) and carbonic anhydrase. Gliosis was most severe in spinal-cord 'white-matter' tracts in the md rat. The most dramatic differences between astrocytes in the md and normal animals were revealed with anti-GFAP and anti-vimentin. Ependymal cells and tanycytes were quite similar in the md and normal CNS. Among the glial-cell antigens, Yb showed the broadest distribution, which included astrocytes, ependymal cells and tanycytes.

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.

Central myelin in the first hybrid mice produced by intercrossing homozygotes of shiverer and myelin-deficient mutants

The first hybrid mice (‘shiverer∗ mld’ mice) produced by intercrossing the homozygotes of the shiverer (BALB/c strain) and mld (MDB/Dt strain) were used for investigating the fine structure of the myelin lamellae, immunoreactive pattern for myelin basic proteins (MBP) and Golgi impregnated images of oligodendrocytes, with special reference to the influence of aging. All of the hybrid mice had an intermediate coat color between the white of the shiverer and black-brown of the mld, and revealed the same neurological symptoms, intention tremor, ataxic behavior, etc., as those of the shiverer and mld. The central myelin lamellae of the ‘shiverer∗ mld’ mouse exhibited the similar characteristics to the shiverer type rather than the mld type from the standpoint of the infrequent occurrence of major dense lines, although they did display a tendency to increase major dense lines with aging like the mld. Observation of the immunohistochemical preparations for MBP showed that immunopositive myelin sheaths were present in the white matter, although they were far more infrequent than those of the mld mutant, probably reflecting the amount of major dense lines. Thus, in the CNS of the ‘shiverer∗ mld’ mouse, the MBP-synthesis was possibly much more disturbed than in the mld mutant, or at least, revealed an intermediate pattern between the mld and shiverer.

Sulfoglucuronyl neolactoglycolipids in adult cerebellum: specific absence in murine mutants with Purkinje cell abnormality.

It is shown here that glycolipids of the sulfoglucuronyl neolacto series (SGGLs) are present in the adult rodent cerebellum. SGGLs were not detected in the cerebellar murine mutants lurcher, Purkinje cell degeneration, and staggerer, in which Purkinje cell loss is the primary defect. SGGLs were present, however, in normal amounts in weaver and reeler mutants, in which there is a major and relatively specific loss of granule cells without obvious deficiency in Purkinje cells. In the myelin-deficient quaking mutant, the expression of SGGLs also was nearly normal. The loss of SGGLs in Purkinje cell-deficient mutants was specific, since most of the major lipids were not affected significantly and only the percentage composition of other lipids, such as sulfatides and gangliosides, was altered in the mutants. These and other results strongly suggest that SGGLs and other glycolipids of the paragloboside family are localized specifically in Purkinje cells and their arbors in the adult cerebellum. This is the first demonstration of the localization of a specific glycolipid and its analogs in a specific cell type in the nervous system.

Stage-specific cell-surface antigens of oligodendrocytes in the peripheral nervous system. Expression during development and regeneration and in myelin-deficient mutants

Monoclonal antibodies to stage-specific cell surface antigens of oligodendrocytes have been used to investigate the expression of antigens 05 through 011 in the peripheral nervous system of the mouse by immunohistology. In the adult sciatic nerve antigens 05 through 09 and 011 were diffusely positive. 010 antigen was not detectable in the peripheral nervous system at any age tested. During development antigens 05, 06 and 07 were first detectable at birth in tracts at the proximal part of the sciatic nerve. At day 2 the whole diameter of the nerve was positive for 05 antigen, while antigens 06 and 07 were detectable only in part of the nerve and antigens 08 and 09 were just about to appear. At day 4 antigen 011 was the last to appear. At day 7 all antigens were strongly detectable throughout the nerve. After transection of adult sciatic nerve expression of antigens 05 through 09 and 011 was studied at the proximal and distal ends of the cut. Three days after transection all antigens were fully detectable in the degenerating myelin and its debris. After 15 days residual debris was still distinctly positive, while Schwann cells in the bands of Büngner were antigen-negative. At approximately two weeks a connecting bridge between proximal and distal ends of the cut nerve had developed, but the 0 antigens were not detectable in this bridge until day 21. At day 42 all antigens were again fully detectable in the regenerating nerve. In hypomyelinating mouse mutants no difference to the normal control littermates was seen in staining pattern and intensity for jimpy and shiverer, while quaking showed an increase in staining intensity for 05 through 08 antigens. In trembler antigens 05, 06 and 07, but not 08, 09 and 011 appeared associated with non-myelin-forming Schwann cells, while the few recognizable myelin-forming Schwann cells expressed all antigens. These observations show that we have characterized 4 new monoclonal antibodies as further reagents to look at developmentally distinct steps in myelination of the peripheral nervous system.