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.     

Structure and Expression of Myelin Basic Protein Gene Sequences in the mld Mutant Mouse: Reiteration and Rearrangement of the Mbp Gene

The mld mutation on chromosome 18 in the mouse is a putative allele of the shiverer (shi) mutation. We have analyzed the structure of myelin basic protein (MBP) gene sequences in mld DNA by restriction mapping of genomic DNA. The results indicate that the mld chromosome carries two copies of the MBP structural gene, one of which is intact and one of which is interrupted. Genetic analysis indicates that the interrupted gene is close to the intact MBP structural gene and cosegregates with the mld mutation. We have also analyzed the levels of MBP polypeptides and MBP-specific mRNA in wild-type, homozygous and heterozygous shiverer and mld mice and in mice carrying both mutations. The results indicate that both shi and mld are cis-acting codominant mutations that cause severely reduced steady state levels of MBP-specific mRNA and MBP polypeptides in the brain. We have analyzed the total number of oligodendrocytes and the number of MBP-positive oligodendrocytes in mld and shi brain primary cultures. In shi cultures, none of the oligodendrocytes expresses MBP. However, in mld cultures, approximately 5% of the oligodendrocytes express MBP. The nature of the "revertant" mld oligodendrocytes is not known.     

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.     

Morphometric Analysis of Normal, Mutant, and Transgenic CNS: Correlation of Myelin Basic Protein Expression to Myelinogenesis

The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)     

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. shimid), 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.     

Gene organization and transcription of duplicated MBP genes of myelin deficient (shi(mld)) mutant mouse

A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712-bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3-kb 5'-flanking regions from respective genes of shi(mld) measured by in vitro run-off assay using HeLa whole-cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed.     

The specificity of the myelin basic protein gene promoter studied in transgenic mice.

The myelin basic proteins (MBPs) are a family of polypeptides that are predominantly expressed in the nervous system where they play a major role in myelination. We have generated four lines of transgenic mice carrying a transgene in which 1.34 kb of the 5'-flanking sequence of the mouse MBP gene was fused upstream of the coding region of the Escherichia coli lac Z gene in order to investigate developmental and tissue-specific expression of the MBP gene. Expression of both the lacZ transgene and the endogenous MBP gene followed a common developmental pattern in mouse brain. Transgene expression was detected in primary oligodendrocytes, but not in type 2 astrocytes. In addition, the lacZ gene product was expressed in epithelial cells of certain nonneural tissues, namely kidney, epididymis, ureter, and seminal vesicles. The ectopic expression of the transgene was associated with the development of DNase I hypersensitive sites at the site of insertion which was found to be within the intron 1 region of the endogenous MBP gene. The results reported here strongly suggest that the 1.34-kb 5'-flanking region of the MBP gene contains cis-regulatory elements that confer developmental regulation of the MBP gene, although this region appears to lack elements that restrict its expression to the nervous system.     

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.     

Developmental Expression of the Myelin Proteolipid Protein and Basic Protein mRNAs in Normal and Dysmyelinating Mutant Mice

Expression of the myelin proteolipid protein (PLP) was examined in the nuclei and polysomes of 12-27-day-old quaking, jimpy, and shiverer mouse brains and in 2-27-day-old normal brains and compared with expression of the myelin basic proteins (MBPs). Northern blots showed the presence of multiple mouse PLP RNAs, the developmental expression of which coincided with myelination. Two major mouse PLP RNAs, 3.5 and 2.6 kilobases in length, were observed in both cytoplasmic polyribosomes and nuclei, and, in addition, a larger 4.6-kilobase PLP RNA was observed in nuclei. Quantitative measurements with slot blot analyses showed that the levels of PLP and MBP RNAs peaked simultaneously at 18 days in nuclei but that maximal levels of PLP RNA lagged behind MBP RNA by several days in the polysomes. The developmental expression of both major classes of myelin protein mRNAs was affected in all three mutants. In shiverer brains, the levels of PLP mRNA in polysomes and nuclei were only 30-55% of control levels after 15 days. Thus, the deletion of a portion of the MBP gene appeared to have a major effect on the expression of the PLP gene in this mutant. In jimpy mice, where the mutation has been shown to involve the PLP gene, expression of MBP mRNA was also severely reduced, to less than 25% of control values. In quaking brains, the expression of each gene followed its own developmental course, different from each other and different from the normal mouse. The extent to which the expression of PLP and MBP was affected by the quaking mutation depended on the age at which it was examined.     

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.     

Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin-Deficient (mld) Mouse Brain

Abstract: Myelin-deficient ( mld ) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10⁻⁶ per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant ( mld/mld , scid/scid ) and single mutant ( mld/mld , + ^scid /+ ^scid ) mice, indicating that the scid factor is not required for MBP gene reactivation in mld . The significance of DNA rearrangement in mammalian development is discussed.     

Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons.

We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.