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.     

Studies on myelin basic protein-specific protein methylase I in various dysmyelinating mutant mice

Jimpy mice are dysmyelinating mutants characterized by producing near normal levels of myelin basic protein (MBP) in the brain but failing to incorporate these proteins into the myelin sheath. In this study, the activity of MBP-specific protein-arginine N-methyltransferase (protein methylase I) was studied in the brains of normal and jimpy mice of different ages. The enzyme activity varied little with age in normal mice but in 18 and 21 days-old homozygous jimpy mice the activity was reduced by 50% and 75% respectively from the level of their normal littermates. Interestingly, however, heterozygous jimpy mice who are phenotypically normal and quaking mice (a similar dysmyelinating mutant) showed unaltered enzyme levels.     

SYNTHESIS OF ETHANOLAMINE PHOSPHOGLYCERIDES BY MICROSOMES FROM THE BRAINS OF JIMPY AND QUAKING MICE

Abstract— —Brains of jimpy and quaking mice are known to be deficient in myelin and alkenylacyl-glycero-phosphorylethanolamines (alkenylacyl-GPE, ethanolamine plasmalogens). Ethanolamine plasmalogen synthetic activity appeared to be normal and ethanolamine phosphotransferase (EC 2.7.8.1) activities are higher in the brain microsomes from jimpy and quaking mice than in their littermate controls when the activities are assayed with alkylacylglycerols and CDP[¹⁴C]ethanolamine. When endogenous diradylglycerols were the substrate, the rate of synthesis of diacyl-GPE was normal but the rate of synthesis of the ether lipids, alkenylacyl-GPE and alkylacyl-GPE, was 33% and 8% below control levels for jimpy brain microsomes and quaking brain microsomes respectively. This difference is probably due to a normal content of diacylglycerols and a deficient content of alkylacylglycerols in the mutant brain microsomes. The apparent alkylacylglycerol deficiencies in the microsomes correspond with the ethanolamine plasmalogen deficiencies in the brains of these mutant mice.     

Alpha hydroxylation of lignoceric acid to cerebronic acid during brain development. Diminished hydroxylase activity in myelin-deficient mouse mutants.

Alpha Hydroxylation of lignoceric acid (n-tetracosanoic acid) to cerebronic acid (2-hydroxylignoceric acid) by postnuclear preparations of brains from developing rat, mouse, and several neurological mouse mutants was studied. The preparations of brains from jimpy and myelin synthesis deficiency (msd) mice were found to synthesize cerebronic acid at less than 10 percent of their control rates, and those from quaking and dilute-lethal approximately 30 and 50 percent, respectively. The apparent low rate of in vitro hydroxylation by brains of the mutant mice appeared to be due to decreased synthesis rather than increased oxidation of cerebronic acid. Mixing experiments eliminated the possibility of an inhibitor in the mutant or an activator in normal animals. The preparations of brains from wabbler-lethal, ducky, and weaver mice showed normal activity. The developmental pattern of the hydroxylase activity was examined in quaking, jimpy, and their control mice. In normal brains the hydroxylase activity was low in the immediate postnatal period, increased sharply between 10 and 20 days after birth, and fell to a low level following maturation of the brain. The hydroxylase activity in quaking mice changed similarly during brain development but at a much reduced level. The brains of jimpy mice had barely detectable hydroxylase activity which changed little with age and reached a peak at about 15 days postpartum. The subnormal hydroxylase activity in brains of quaking mice and the near absence in brains of jimpy and msd mice correlate with the observations that myelin deficiency is more severe in jimpy and msd than in quaking. These results suggest a close association of the synthesis of cerebronic acid with the synthesis of the characteristic myelin lipid that is cerebroside (N-acyl sphingosine beta-D-galactoside).     

Synthesis of the myelin proteolipid protein in the developing mouse brain

Mice ranging in age from 16 to 44 days were injected intracerebrally with ³H-leucine, and incorporation into total brain proteolipids and the myelin proteolipid protein was measured. All proteolipids were isolated from whole brain by ether precipitation and separated into their individual components by SDS polyacrylamide gel electrophoresis. Two major proteolipids with apparent molecular weights of 20,700 and 25,400 were observed in these preparations, and their proportion increased over the developmental period examined. A Ferguson plot analysis comparing these proteins with those of isolated myelin showed that the 25,400-dalton proteolipid component from whole brain was the myelin proteolipid protein. Rates of incorporation of ³H-leucine into total brain proteolipids peaked at 22 days of age. Synthesis of the myelin proteolipid protein increased rapidly to a maximum value at 22 days and decreased rather slowly until at 44 days it was about 83% of its maximum rate of synthesis. The data indicate that the developmental pattern of synthesis of the myelin proteolipid protein is unlike that of the myelin basic proteins. Synthesis of the major myelin proteins is developmentally asynchronous in that peak synthesis of the myelin proteolipid appears to occur several days later than the basic proteins. In addition, it maintains its maximum rate of synthesis over a longer period of time than do the basic proteins.     

PROTEOLIPID PROTEIN: SYNTHESIS AND ASSEMBLY INTO QUAKING MOUSE MYELIN

The incorporation of radioactive glycine into the major myelin proteolipid protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. In a typical experiment, four Quaking mice and four littermate controls were injected intracranially with 250 μCi [2-³H]glycine and 25 μCi [U-¹⁴C]glycine respectively. Three hours later, the eight mice were killed and their brains combined. Equivalent portions were taken for (1) chloroform-methanol (2:1) extraction followed by ether precipitation of proteolipid from the brain and (2) myelin preparation. The ^3H/¹⁴C ratios for the microsomes:, the major myelin proteolipid as well as the other non-myelin proteolipids extracted from whole brain was approx 3.0. while the ³H/¹⁴C ratio for proteolipid protein in myelin was near 0.4. These findings were consistent for ages studied between 18 and 90 days. The results indicate that the synthesis of the major myelin proteolipid protein in the whole brain of Quaking mouse, as seen previously in our studies on basic protein, proceeds at a normal rate relative to microsomes but its incorporation into myelin is depressed. A working hypothesis of myelin membrane assembly is presented to account for the defect in the incorporation of these proteins into Quaking myelin.     

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.     

PLASMALOGENASE ACTIVITIES IN THE BRAINS OF JIMPY AND QUAKING MICE

The activity of plasmalogenase, which hydrolyzes the vinyl ether linkage of the plasmalogen molecule, increased markedly in control mouse brains during the period of most active myelin deposition. Only a slight increase in plasmalogenase activity was found in brains from jimpy mice. At all ages studied, the jimpy mouse brains had less plasmalogenase activity than the littermate control brains and this disparity increased with increasing age. By 25 days of age the jimpy brains contained only 43% of the activity observed in control brains. Adult quaking mouse brains also had significantly less plasmalogenase activity when compared to littermate controls. Thus, the plasmalogenase activities correlate well with the degree of myelination.     

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands. Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.     

An AG----GG transition at a splice site in the myelin proteolipid protein gene in jimpy mice results in the removal of an exon

The myelin proteolipid protein gene was characterized in jimpy mice to identify the specific mutation that produces dysmyelination, oligodendrocyte cell death, and death of the animal by 30 days of age. Exon 5 and flanking intron segments were isolated from jimpy proteolipid protein genomic clones and sequenced. A single nucleotide difference was noted between the normal and jimpy proteolipid protein genes: the conversion of an AG/GT to a GG/GT in the splice acceptor signal preceding exon 5, which apparently destroys the splice signal. Thus, exon 5 of the mouse myelin proteolipid protein gene is skipped during the processing of mRNA, producing a shortened proteolipid protein mRNA.     

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.     

A Combination of PLP and DM20 Transgenes Promotes Partial Myelination in the Jimpy Mouse

Abstract: Mutations in the myelin proteolipid protein (PLP) gene, such as that found in the jimpy mouse, result in an abnormal structure of the myelin, severe dysmyelination, and a reduction in the number of mature oligodendrocytes. To examine the functions of the two alternatively spliced isoforms of proteolipid protein, transgenic mice were generated that express either PLP or DM20 cDNAs placed under control of the PLP upstream regulatory region. The transgenes were bred into jimpy mice, and the effect of the transgenes on the dysmyelinating phenotype was analyzed. Neither the PLP transgene nor the DM20 transgene alone had an effect on myelination in the jimpy mice. Combining the two transgenes substantially increased the number of myelinated axons, suggesting that the two alternatively spliced products of the PLP locus perform distinct functions in oligodendrocytes. The enhanced myelination was not sufficient, however, for completely correcting the dysmyelinating phenotype of the jimpy mice, nor was it accompanied by the restoration of normal levels of myelin gene expression. The inability to rescue the jimpy phenotype is most likely attributable to a dominant negative action of the abnormal proteolipid proteins present in jimpy mice. These results demonstrate the complexity of proteolipid protein function in myelination.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

A spin-label study of sciatic nerves from quaking, jimpy and trembler mice.

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant (T) between 0 degrees C and 50 degrees C. Within this range of temperatures, we obtained identical values of 2 T for nerves from normal and jimpy mice, whereas 2 T was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice. The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.     

In vitro acylation of myelin PLP and DM-20 in the quaking mouse brain

Both proteolipid proteins (PLP) and DM-20 were found to be present by the immunoblot technique in myelin isolated from quaking mouse brain; however, the relative concentration of these proteins in myelin from quaking brain was substantially reduced when compared to the control. Brain slices from littermate control and quaking mice were incubated with [³H]palmitic acid to determine the incorporation of fatty acid into myelin proteolipid proteins. Fluorography of gels containing myelin proteins from control and quaking mice brain revealed that both PLP and DM-20 were acylated. The incorporation of [³H]palmitic acid into quaking myelin PLP and DM-20 was reduced by 75% and 20% respectively of those in control brain. The significance of differential acylation of quaking myelin PLP and DM-20 is discussed with respect to availability of non-acylated pools of proteolipid proteins and the activities of acylating enzymes.     

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.     

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.     

Biochemical Abnormalities in Spinal Cord Myelin and CNS Homogenates in Heterozygotes Affected by the Shiverer Mutation

Myelin was purified from the spinal cords of normal mice and mice heterozygous for the shiverer mutation, and measurements were made of the major myelin proteins and lipids and the specific activities of three myelin-associated enzymes. The myelin purified from the spinal cords of the heterozygotes (shi/+) was deficient by 30-40% in yield and had an apparently unique composition. In particular, when compared with normal mouse spinal cord myelin, there were more high-molecular-weight protein, less myelin basic protein, a higher protein-to-lipid ratio, and higher specific activities of 2',3'-cyclic nucleotide-3'-phosphohydrolase (EC 3.1.4.37) and carbonic anhydrase (EC 4.2.1.1) in the myelin purified from the shi/+ animals. These abnormalities were reflected in the composition of shi/+ whole spinal cord, where the protein-to-lipid ratio was intermediate between the respective values for +/+ and shi/shi spinal cords. Whole brains from shi/+ mice showed deficiencies in galactocerebroside and galactocerebroside sulfate and an increase in total phospholipid, and the lipid composition in the brains of the shi/shi mice was similar to that reported for another dysmyelinating mutant, quaking. The findings provide the first values for the lipids in normal mouse spinal cord myelin and show that heterozygotes are affected by the shiverer mutation. The observations imply that there can be considerable deviation from the normal CNS myelin content and composition without apparent qualitative morphological abnormalities or loss of function and that the amount of myelin basic protein available during myelination may influence the incorporation of other constituents into the myelin membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE COMPOSITION OF MYELIN FROM THE MUTANT MOUSE ''QUAKING''

Abstract— Myelin was isolated from the brains of adult Quaking mice, a mutant showing a deficiency of myelin in the central nervous system, and normal controls. The mutant myelin was found to have a higher flotation density than that of the control and showed marked differences in lipid composition. The myelin from Quaking mice was found to be deficient in cerebroside and ethanolamine phospholipid. Acrylamide gel electrophoresis of total myelin protein demonstrated a pronounced deficiency of proteolipid protein. The activity of cyclic 2',3'-AMP phosphohydrolase was normal.