Myelin Basic Protein Synthesis Research Articles

Translational regulation of myelin basic protein synthesis

Synthesis of the mouse myelin basic proteins (MBPs) was studied in reticulocyte lysates programmed with brain mRNA in the presence or absence of brain factors. Addition of brain factors to the lysates increased the incorporation of [35S]methionine into total TCA-precipitable protein by a factor of 6-9, and the majority of this stimulation was found to be due to initiation factors. Although brain factors increased total protein synthesis, the percentage of MBP synthesis was reduced from 4.3% of the total counts incorporated (in the absence of brain factors) to 1.4% (in their presence). Increasing the concentration of brain mRNA in the lysates also reduced the relative levels of MBP synthesis. These results suggested the MBP mRNAs, as a group, were less efficiently initiated than most brain mRNAs. An analysis of the nucleotide sequence flanking the initiator codon of the MBP mRNAs indicates the presence of a second AUG codon 5 bases upstream, immediately followed by a termination codon, which may provide a structural explanation for the poorer initiation efficiency of the MBP mRNAs. Further analysis of the synthesis of the individual MBPs in the presence or absence of inhibitors of initiation (7-methylguanosine triphosphate and aurintricarboxylic acid) and elongation (anisomycin and emetine) indicated than the 14-kD MBP mRNA was less efficiently translated than the other MBP mRNAs. Synthesis of the 14-kD MBP was more strongly inhibited by the initiation inhibitors than the other MBP mRNAs, and synthesis of the 14-kD MBP was increased relative to the other MBPs in the presence of elongation inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligodendrocyte Adhesion Activates Protein Kinase C-Mediated Phosphorylation of Myelin Basic Protein

When isolated adult oligodendrocytes adhere to a substratum myelinogenesis occurs. Investigation of the mechanism by which this happens indicated that the oligodendrocyte-substratum interaction activated protein kinase C-dependent phosphorylation of myelin basic protein and promoted the synthesis of myelin basic protein. In addition, when agents that activate protein kinase C (second messenger diacylglycerol or a tumor-promoting phorbol ester) were added to nonattached oligodendrocytes, they mimicked the influence of the substratum by inducing phosphorylation of myelin basic protein; and reagents that increase cellular adenosine 3', 5'-monophosphate (cyclic AMP) inhibited phosphorylation of myelin basic protein. Thus, at least in vitro, the interaction between oligodendrocytes and the substratum may mediate myelinogenic events, and phosphorylation of myelin basic protein may be an early requirement in the sequence of steps that ultimately results in myelin formation.

Studies on myelin-basic-protein methylation during mouse brain development

The synthesis and methylation in vivo of myelin basic protein (MBP) during the mouse brain development has been investigated. When mice ranging in age from 13 to 60 days were injected intracerebrally with L-[methyl-3H]methionine, the incorporation of radioactivity into MBP isolated from youngest brain was found to be the highest and declined progressively in mature brains. This pattern of radioactivity incorporation was inversely correlated with the total amount of MBP in the brains, suggesting a higher ratio of MBP methylation to synthesis in younger brain. To differentiate the relative rate of protein synthesis and methylation, animals were given intracerebral injections of a L-[methyl-3H]methionine and L-[35S]methionine mixture and the ratio of 3H/35S (methylation index) was determined. The ratios in the isolated MBP fractions were higher than those of 'acid extracts' and 'breakthrough' fractions, with a maximal ratio in the youngest brain. This high ratio was well correlated with the higher protein methylase I (PMI) activity in younger brains. The MBP fractions were further separated on SDS/polyacrylamide-gel electrophoresis into several species with apparent Mr ranging from 32,400 to 14,500. The results indicated that each protein species accumulated at a characteristic rate as a function of age. The high-Mr (32,400) species was predominant in younger brain, whereas the smaller MBP was the major species in older brain tissue. The importance of this developmental pattern of MBP synthesis and methylation is discussed in relation to PMI activity.

Mice heterozygous for the mld mutation have intermediate levels of myelin basic protein mRNA and its translation products

Myelin-deficiency ( mld) is an autosomal recessive mutation in mice exhibiting a severe deficit in the synthesis of myelin basic protein (MBP). In order to understand the mechanisms involved in the regulation of MBP synthesis in the mld mutation, we examined the amount of MBP and MBP-specific mRNA in control, heterozygous and homozygous mld brains. In vitro translation of poly(A) + RNA in a cell-free system, in situ hybridization, and filter hybridization with a radiolabelled probe pMBP-1 after dot or Northern blotting were used in this study. The levels of MBP and MBP-specific mRNA were very low but detectable in mld homozygotes, and intermediate in heterozygotes. MBP specific mRNA from mutants, and its translation products, were of normal size. These results show that the mld mutation is expressed co-dominantly in heterozygotes and affects a cis-acting regulatory element controlling the MBP gene.

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

Expression of myelin proteins was studied in the brains of 21-day-old normal mice and three dysmyelinating mutants-jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell-free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21-day-old quaking brain homogenates showed a reduction in the steady-state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21-day jimpy brain homogenates revealed no appreciable steady-state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.

Co-culture study of rat neuron-glial interaction: Evidence of neuronal influence on myelination

Organotypic cultures of newborn rat brains were exposed to the neurotoxin kainic acid or the DNA synthesis inhibitor arabinoside C. The cultures were subsequently co-cultured and the myelination-related enzymatic activities, such as 2′,3′-cyclic nucleotide phosphohydrolase and uridine diphosphate-galactoseceramide galactosyl transferase, were determined under various culture conditions. The newly formed myelin basic protein in the cultured brain tissue was determined by the radioimmunoprecipitation method. The myelination-related enzymatic activities and the synthesis and accumulation of myelin basic protein in the co-cultured brain tissue were found compatible to the control cultures which were not exposed to either drug. The cultures which had been treated with either drug, but not subsequently co-cultured, were found to have decreased enzymatic activities and myelin basic protein synthesis. The experimental data suggest that myelinogenesis requires an interaction between functional neurons and oligodendroglial cells and further supports the hypothesis that the neuron exerts a regulatory effect on the glial myelination mechanism.

Murine leukodystrophies as tools to study myelinogenesis in normal and pathological conditions.

Myelinogenesis is controlled by several genes. Therefore, the study of mutations affecting myelination should provide better understanding of the assembly and maintenance of myelin, and in the case of similitude with human diseases, a direct insight into the pathogenesis of these diseases. Murine mutants can be bred readily and sequential analyses allow an examination of the dynamic processes of myelination. In this study, we have selected certain aspects of four leukodystrophies in mice. The precise mechanisms leading to the important myelin deficit observed in the jimpy mutation, a sex-linked recessive trait, are still not completely elucidated. Our results show in jimpy mice severe astrocytic alterations prior to myelin formation. Therefore, abnormalities of oligodendrocytes and possibly axons could be a secondary phenomenon. Nevertheless, a defect involving neuroglia precursor cells cannot be ruled out. At the present time, Pelizaeus-Merzbacher is a disease in which mainly oligodendrocytes appear to be altered. In consequence, our findings in jimpy mice do not support the present contention that this mutation represents a model for Pelizaeus-Merzbacher disease. We propose that the jimpy mutation could be invaluable to study cell-cell interactions at an early stage of myelinogenesis. Interest in the myelin deficient (mld) mutant derives from the specific lack of one of the major myelin constituents, myelin basic protein, and the concomitant absence of the major electron dense line during the active phase of myelin deposition in the CNS. Our present knowledge points out a defect of gene regulation involving myelin basic protein synthesis. The absence of this extrinsic membrane component made it possible to catch a glimpse of its role in the formation and maintenance of the complex structure of the myelin sheath in the CNS. Quaking mice have been considered to represent an arrest of myelinogenesis. This commonly accepted view has to be reconsidered at the light of recent morphological and biochemical findings. An abnormal processing of the myelin-associated glycoprotein could prevent the normal compaction of myelin until the animals reach adulthood. Twitcher is characterized by extensive central and peripheral demyelination and the presence of intracellular inclusions in macrophages similar to human globoid cell leukodystrophy. In both human and murine diseases, a deficiency of galactosylceramidase activity was demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

MRNA-directed cell-free myelin basic protein synthesis: No evidence of neuronal control

The specific mRNA of myelin basic protein (MBP) was isolated from active myelinating rat brains. The isolated mRNA was able to direct the synthesis of immunochemically detectable MBP in a wheat-germ cell free protein synthesis system. There was, however, no evidence of neuronal translational control of the in vitro MBP synthesis kinetics found within the experimental parameters. The possibility of neuronal control of MBP gene expression is therefore postulated likely to be at the transcriptional level.

Cell-free synthesis of the myelin basic proteins in a wheat germ system programmed with brain messenger RNA.

Poly A(+) messenger RNA (mRNA) was isolated from the brains of 3-week-old mice and translated in a cell-free system derived from wheat germ. Maximal stimulation of the system by brain mRNA was observed at a relatively low K+ concentration (45 mM) and low mRNA concentration (1-10 microgram/ml). The translational system was dependent on an energy-generating system and stimulated by the addition of spermidine and transfer RNA. Under optimal conditions, incorporation was linear for almost 45 min, but the overall stimulation with brain mRNA was relatively low (about twofold). In spite of the low stimulation, analysis of the translation products indicated that in the presence of brain mRNA polypeptides which co-chromatographed and co-electrophoresed with the two mouse myelin basic proteins could be detected. In control experiments with liver poly A(+) mRNA, which stimulated the translational system to a greater extent than brain mRNA, no such polypeptides could be detected. In this system the ratio of synthesis of small myelin basic protein to large myelin basic protein was found to be about 4.0, which correlates well with that found in vivo.

Synthesis of myelin basic proteins in the developing mouse brain

Mice ranging in age from 14 to 39 days were injected intracerebrally with [ 3H]lysine and rates of incorporation of the isotope were measured into total trichloroacetic acid-precipitable protein and purified myelin basic proteins (MBPs). MBPs were isolated by O-(carboxymethyl)-cellulose chromatography of pH 3 extracts prepared from chloroform-methanol insoluble residues of whole brains. The MBPs prepared in this fashion were further separated by polyacrylamide gel electrophoresis. The gels were sliced and the radioactivity incorporated into each of the two proteins was determined. Analysis of the rates of synthesis of the two basic proteins (using a 2-h labeling period) as a function of age revealed that synthesis of both proteins appeared to peak at about 18 days of age in the mouse. These data suggest that the maximum rate of MBP synthesis coincides with the age of maximal myelin deposition in the mouse. Furthermore the relative rates of synthesis of L and S changed considerably over the developmental period examined. It was observed that the ratio of the rates of synthesis of the small:large basic protein (S/L) increased by approximately 50% between 2 and 4 weeks and declined thereafter. Throughout the developmental period examined, however, the small basic protein appeared to be synthesized at a greater rate than the large protein. The latter data are consistent with previous observations by us and other workers that mouse and rat myelin becomes increasingly enriched in the small relative to the large basic protein with maturation of the membrane.

Immunochemical measurement of myelin basic protein in developing rat brain: An index of myelin synthesis

The initial time and rate of myelin basic protein synthesis in neural tissues of the rat have been measured from birth to 120 days. The protein was quantitated by a radioimmunoassay directly applied to unfractionated cerebrum, cerebellum, olfactory bulb, midbrain, brain stem, optic and trigeminal nerve, and areas of the spinal cord. Because the protein is a specific myelin constituent and its appearance correlates precisely with the synthesis of myelin lipids, the data in this report can be interpreted in terms of myelin synthesis and oligodendrocyte activity. The results show striking heterogeneity in the initial time and rate of myelin synthesis in neural tissue.

Glial cells and the central myelin sheath.

Glial cells and the central myelin sheath.