Expression of Myelin Basic Protein Genes in Several Dysmyelinating Mouse Mutants During Early Postnatal Brain Development

Northern blot and "dot" blot analyses using a myelin basic protein (MBP) specific cDNA probe and in vitro translation techniques were utilized to estimate the relative levels of myelin basic protein messenger RNA (mRNA) in the brains of C57BL/6J control mice, three dysmyelinating mutants (qk/qk, jp/Y, and shi/shi), and three heterozygote controls (qk/+, jp/+, and shi+) during early postnatal development. In general, the MBP mRNA levels measured directly by Northern blot and "dot" blot analyses correlated well with the indirect in vitro translation measurements. The Northern blots indicated that the size of MBP mRNAs in quaking and jimpy brain polysomes appeared to be similar to controls. Very low levels of MBP mRNAs were observed in shi/shi brain polyribosomes throughout early postnatal development. Compared to C57BL/6J controls, accumulation of MBP mRNAs in qk/qk and qk/+ brain polyribosomes was delayed by several days. That is, whereas MBP mRNA levels were below normal between 12 and 18 days, normal levels of message had accumulated in both qk/qk and qk/+ brain polyribosomes by 21 days. Furthermore, normal levels of MBP mRNAs were observed to be maintained until at least 27 days. MBP mRNA levels remained well below control levels in jp/Y brain polyribosomes throughout early postnatal development. The levels did, however, fluctuate slightly and peaked at 15 days in both jp/Y and jp/+ brains, 3 days earlier than in normal mice. Thus, it appears that jimpy and quaking mice exhibit developmental patterns of MBP expression different from each other and from C57BL/6J control mice.     

Abnormal Expression of the Myelin-Associated Glycoprotein in the Central Nervous System of Dysmyelinating Mutant Mice

Total cytoplasmic brain RNA was isolated at two different ages from three neurological mutant mice (qk/qk, jp/Y, and shi/shi) and their apparently normal littermates. This RNA was translated in vitro in a rabbit reticulocyte lysate system. Myelin-associated glycoprotein (MAG)-related polypeptides were immunoprecipitated from equal amounts of total translation products derived from mRNA of mutant animals, normal littermates, or control animals. The developmentally regulated synthesis of MAG polypeptides was compared among the mutants and normal animals. mRNA from qk/qk brains synthesized an overabundance of p67MAG (five- to sevenfold) which may be compensation for a decreased synthesis of p72MAG. mRNA from jp/Y brains synthesized less than 10% of normal amounts of both MAG polypeptides. The quantity of MAG synthesized by 15-day shi/shi brain mRNA was slightly decreased compared with normal brain mRNA but the quantity of MAG synthesized by adult shi/shi brain mRNA was normal. No apparent differences were detected in the sizes of the MAG polypeptides synthesized by any of the mutants studied. The data suggest that the genetic defect in qk/qk mutants directly or indirectly affects the coordinated developmental regulation of MAG polypeptide synthesis leading to an overabundance of the MAG polypeptide that is normally found in older animals. The jp/Y mutation appears to affect general myelin protein synthesis. Finally, shi/shi mutants may have a delayed synthesis of MAG. The data are discussed in the light of recent observations concerning the synthesis of myelin proteins and their proposed role in myelin assembly.     

Cell-Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice

Total polyribosomes were isolated from the brains of 16-20 day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shil/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 +/- 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and less than 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an "assembly" or "synthesis" defect.     

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype. © 1996 John Wiley & Sons, Inc.     

Nuclear retention of MBP mRNAs in the quaking viable mice

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.     

Brain DNA metabolism in myelin deficient mutant jp,jpmsd and qk mice

We studied metabolism of brain DNA in three myelin deficient mutants qk, jp and jp^msd mice. The DNA content, the in vivo incorporation of [¹⁴C]thymidine in DNA and the activity of acid DNase in tissues (cerebellum and cerebrum) from normal littermates and affected mice were compared. The results showed that neither the DNA content, the incorporation of [¹⁴C]thymidine in DNA nor the activity of acid DNase in brain were altered in qk affected mice. In jp^msd mice, however, the DNA content as well as the incorpation of thymidine in DNA were reduced in both cerebellum and cerebrum, but the activity of acid DNase was reduced in cerebrum only. In jp mice, although the DNA content was reduced in both cerebellum and cerebrum, the incorporation of thymidine in DNA and the activity of acid DNase were reduced in cerebrum only. The data suggest a) that in qk mutants DNA metabolism and hence cell (glial) proliferation is not affected; b) that in jp^msd mutants DNA synthesis, and thus the cell proliferation is reduced in cerebellum as well as in cerebrum of the affected mice and c) that in jp mutants the synthesis of DNA and the cell proliferation is reduced in cerebrum but not in cerebellum.     

The Effect of the Shiverer Mutation on Myelin Basic Protein Expression in Homozygous and Heterozygous Mouse Brain

We report (a) that the shiverer mutation has pleiotropic phenotypic effects on myelin basic protein expression in the CNS of homozygous (shi/shi) mice and (b) that each of the effects of the shiverer allele is expressed co-dominantly with the wild-type allele in heterozygous (+/shi) animals. First, the total amount of myelin basic protein, as determined by radioimmunoassay, that accumulates in the CNS is approximately 0.1% of the wild-type amount in shi/shi animals and approximately 50% in +/shi animals. Second, the four major forms of myelin basic protein, with molecular weights of 21,500, 18,500, 17,000, and 14,000, that are present in wild-type mouse CNS are undetectable in either whole brain or purified myelin of shi/shi animals, and each of the four proteins is reduced commensurately in brain and myelin of +/shi animals. Third, the small amount of myelin basic protein-related material that does accumulate in the shi/shi brain consists of several polypeptides, with molecular weights ranging from 25,000 to 100,000, the pattern of which is different from that found in wild-type brain. The pattern of myelin basic protein-related polypeptides in +/shi brain is a composite of the wild type and the shiverer mutant. Fourth, messenger RNA from shi/shi brain, when translated in vitro, encodes a set of myelin basic protein-related polypeptides qualitatively similar to that encoded by wild-type messenger RNA, except that the 18,500 and 14,000 translation products are greatly reduced, while other myelin basic protein-related translation products are spared. The pattern of myelin basic protein-related translation products for +/shi messenger RNA is intermediate between the patterns for +/+ and shi/shi messenger RNAs. The results suggest that the genetic lesion in the shiverer mutation impinges on the structural gene (or genes) encoding myelin basic protein or on a cis-acting regulatory element controlling that gene (or genes).     

Effect of the Jimpy Mutation on Expression of Myelin Proteins in Heterozygous and Hemizygous Mouse Brain

The levels of myelin basic protein, proteolipid protein, and 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) in cerebral hemispheres of wild-type, heterozygous jp/+, and hemizygous jp/Y mice of different ages were determined by radioimmunoassay and immunoblotting. In jp/Y brain the level of myelin basic protein was 8% that of wild-type at all ages. All forms of the protein were reduced although the 21.5K Mr form was relatively spared at early ages compared to the 18.5K, 17K, and 14K Mr forms. The level of 2',3'-cyclic nucleotide 3'-phosphohydrolase was 8% that of wild-type at all ages, and proteolipid protein was undetectable at any age. These results are consistent with the hypothesis that the jimpy mutation blocks myelin morphogenesis subsequent to incorporation of 21.5K Mr myelin basic protein but prior to incorporation of proteolipid protein. In jp/+ brain the levels of the three proteins were reduced commensurately to 60-70% those of wild-type. The deficit was apparent as early as 10 days after birth and remained proportionately constant throughout development. These results suggest that in jp/+ mice, X-chromosome inactivation produces a mosaic population of functionally wild-type and functionally jimpy oligodendrocytes. The former elaborate normal amounts of myelin but do not completely compensate for the myelin deficit due to the latter.     

Myelin Proteolipid Protein Gene Expression in Jimpy and Jimpymsd Mice

Proteolipid protein (PLP) gene expression was studied in the dysmyelinating mouse mutant jimpy(msd) (jpmsd; myelin synthesis deficient) and compared with that in wild-type mice and the allelic mutant, jimpy (jp). Southern analyses of genomic DNA from jpmsd mice revealed no major rearrangements of the PLP gene relative to the wild-type mouse PLP gene. PLP-specific mRNA levels were significantly reduced in these mutant mice, although both the 3.2- and 2.4-kilobase PLP-specific mRNAs were seen. Also, no size differences in either PLP or DM20 mRNAs were found by S1 nuclease assays of brain RNA from either jpmsd or wild-type mice. Both PLP and DM20 protein were detectable at low levels in jpmsd brain homogenates, and these proteins comigrated with PLP and DM20 protein from normal mice. Western analyses showed an altered PLP:DM20 ratio in jpmsd mice relative to wild-type mice; DM20 levels exceeded PLP levels. It is surprising that a similar pattern of expression was seen in normal mice at less than 10 days of age: DM20 protein expression preceding PLP expression. Thus, jpmsd mice are capable of synthesizing normal PLP and DM20 protein; however, the PLP gene defect has affected the normal developmental pattern of expression for these two proteins.     

Developmental regulation of myelin basic protein expression in mouse brain

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.     

Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

The selective RNA-binding protein QKI is essential for myelination in the central nervous system (CNS). QKI belongs to the family of signal transduction activators of RNA (STARs), characteristic of binding RNA and signaling molecules, therefore is postulated to regulate RNA homeostasis in response to developmental signals. Here we report that QKI acts downstream of the Src family protein tyrosine kinases (Src-PTKs) during CNS myelination. QKI selectively interacted with the mRNA encoding the myelin basic protein (MBP). Such interaction stabilized MBP mRNA and was required for the rapid accumulation of MBP mRNA during active myelinogenesis. We found that the interaction between QKI and MBP mRNA was negatively regulated by Src-PTK-dependent phosphorylation of QKI. During early myelin development, tyrosine phosphorylation of QKI in the developing myelin drastically declined, presumably leading to enhanced interactions between QKI and MBP mRNA, which was associated with the rapid accumulation of MBP mRNA and accelerated myelin production. Therefore, developmental regulation of Src-PTK-dependent tyrosine phosphorylation of QKI suggests a novel mechanism for accelerating CNS myelinogenesis via regulating mRNA metabolism.     

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)     

A novel metalloproteinase associated with brain myelin membranes. Isolation and characterization

Brain myelin membrane preparations contain a metalloproteinase activity which degrades myelin basic protein (MBP). The activity was associated with lentil lectin-binding glycoproteins solubilized from myelin and could be detected in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS). The metalloproteinase represented about 5% of this glycoprotein fraction and was isolated from it by chromatography on DEAE-Sephacel, CM-Sepharose, and Superose 6. The proteinase had an apparent relative molecular weight (Mr) of approximately 58,000 both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr value was unaffected by the presence of reducing agents but was diminished to about 52,000 by treating the proteinase with endoglycosidase F. The purified proteinase cleaved many bonds in MBP but did not generate trichloroacetic acid-soluble products. Two major polypeptides, putatively MBP1-73 and MBP74-170, were prominent in digests of MBP by either the purified enzyme or myelin membranes. The proteinase was active between pH 7 and 9 and was inhibited by phenanthroline and dithiothreitol but not phosphoramidon or inhibitors of serine or cysteine proteinases. Histones, but not azocasein, also served as substrates for the proteinase. From its enzymic and molecular characteristics the myelin-derived metalloproteinase appears distinct from previously described enzymes.     

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.     

Storage protein precursor polypeptides in cotyledons of Pisum sativum L. Identification of, and isolation of a cDNA clone for, an 80000-Mr legumin-related polypeptide

An 80 000-Mr polypeptide, which bound to anti-legumin IgG, was detected among labelled polypeptides from cotyledons at late stages of development. When poly(A)-containing RNA from similar cotyledons was translated in a cell-free protein-synthesizing system, an 80 000-Mr polypeptide was also detected. Immunoprecipitation of translation products with anti-legumin IgG showed that, in addition to the major legumin precursor polypeptides of Mr approximately 60 000, the 80 000-Mr polypeptide was specifically immunoprecipitated. A cDNA clone, pCD32, was found to select an RNA coding for an 80 000-Mr polypeptide in hybrid-selection experiments. Additional minor polypeptides of Mr 63 000 and 65 000 were present in translation products of RNA selected by pCD32; all three polypeptides were immunoprecipitated by anti-legumin IgG. Thermal elution of RNAs bound to pCD32 showed that the affinity of pCD32 to the RNA coding for the 80 000-Mr polypeptide was greater than to the RNAs coding for the 63 000-Mr and 65 000-Mr polypeptides. In similar hybrid-selection experiments, another cDNA clone, pCD40, selected RNAs coding predominantly for polypeptides of Mr 63 000 and 65 000. A minor polypeptide of Mr 80 000 was also detected among these products; again all three polypeptides were immunoprecipitated by anti-legumin IgG. Peptide mapping revealed close similarities between the 80000-Mr polypeptide and the 63 000-Mr/65 000-Mr polypeptides obtained by translation of RNAs selected by pCD32. There were similarities also between maps obtained from translation products of RNA selected by pCD32 and those obtained from anti-legumin IgG immunoprecipitates of total translation products of poly(A)-containing RNA.     

The presence of 17K Mr protein,a major specific substrate for kinase C,found in the Triton-insoluble fraction of synaptosome prepared from rat brain

Cytoskeletal preparation obtained from synaptosome fractions of rat cerebrum contained the activity of kinase C, which phosphorylated 17K Mr protein endogenous to the preparation. The kinase C activity associated with the synaptosome cytoskeletons is greater in the cerebellum and hippocampus than in the cerebrum. The enhancement rates of phosphorylation of the 17K Mr protein were 293%, 544%, and 526% in the Triton X-100-insoluble fractions of synaptosomes prepared from cerebral cortex, hippocampus, and cerebellum, respectively. The 17K Mr protein was distinct from myelin basic protein (MBP) for the following reasons: 1) The electrophoretic mobility of the protein was slightly smaller than that of major MBP of rat in the polyacrylamide gel of 10–20% linear gradient, and the protein was not contained in the purified rat myelin. 2) The isoelectric point of the protein was in neutral range, whereas that of MBP was in alkaline one. 3) The 17K Mr protein did not cross-react with anti-MBP antibody. The protein was shown to be a major substrate contained in the cytoskeletal preparation of synaptosome obtained from cerebrum except for contaminating MBP. Only serine residue of the 17K Mr protein was phosphorylated by the kinase C endogenous to the preparation. The results suggest strongly that the synaptic role of protein kinase C through phosphorylation of the 17K Mr protein.Abbreviations used EGTA ethyleneglycol-bis(-aminoethyl ether) - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - MBP myelin basic protein - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPM synaptic plasma membrane