Myelin-Deficient Rat: Analysis of Myelin Proteins

Myelin basic protein (BP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity were quantitated in the brains and spinal cords of normal and myelin-deficient (md) rats at 8, 12, 18, and 25 days of age. The levels of BP, MAG, and CNP in 25-day-old md brain were 1.1, 1.8, and 11% of those in controls, respectively. In spinal cord, the levels were higher, at 9, 15, and 12% of control values, respectively. Although BP content in the mutant rats was a lower percentage of the control level than MAG and CNPase contents at all ages, the absolute level of BP increased steadily between 8 and 25 days of age in both brain and spinal cord, whereas there was little change in the amounts of MAG and CNPase during this period. Immunoblotting analysis did not reveal an increased apparent Mr for MAG, as has been observed in quaking and trembler mice. There was little difference in the relative distributions of the 14K, 17K, 18.5K, and 21.5K forms of BP between control and md rat spinal cord homogenates at the ages examined. PLP content was reduced more than that of the other proteins in the md mutants, because it could not be detected by a technique capable of detecting 0.2% of the control brain level and 0.1% of control spinal cord level. This suggests that the expression of PLP may be preferentially affected in the md mutation.     

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.     

Jimpy Mice: Quantitation of Myelin-Associated Glycoprotein and Other Proteins

Myelin-associated glycoprotein (MAG), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity, myelin basic protein (BP), and proteolipid protein (PLP) were quantitated in the brains of 20-day-old Jimpy and control mice. The levels of MAG, CNPase, and BP in Jimpy brains were 5.3%, 9.7%, and 1.9% of those in control brains, respectively. Immunoblotting analysis did not reveal an increased apparent Mr for MAG in the Jimpy mouse, as has been observed in some other hypomyelinating murine mutants. PLP was reduced more than the other proteins, as it was not detected by an immunoblotting technique that was capable of detecting 0.5% of the control level.     

Effects of Congenital Infection of Sheep with Border Disease Virus on Myelin Proteins

Abstract— Border disease (BD) of sheep is caused by a virus in the genus Pestivirus that results in decreased myelination throughout the CMS when acquired congenitally. Pregnant ewes were inoculated with BD virus at 50 days of gestation, and myelin proteins were quantified in several regions of the CNS during prenatal and postnatal development of infected lambs for comparison with age-matched controls. Newborn field-infected lambs were also examined. Myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were measured by densitometric scanning of western blots. Deficiencies in the myelin proteins were detected as early as 116 days of gestation, and the deficiencies of myelin proteins were most pronounced in the cerebellum at all ages examined. PLP and MBP increased from 10–30% of normal in cerebellar white matter at birth to 40–60% of normal at 6 months, suggesting some catch-up in the amount of compact myelin with development. MAG and CNP were between 70 and 80% of control levels in the cerebellum at birth and at 6 months. Similar results were obtained for the corpus callosum and spinal cord of infected lambs, but the deficiencies of myelin proteins were not as great. A common finding in all regions examined was that MBP and PLP were reduced more than MAG and CNP. This is probably explained by a greater deficit of compact myelin, in which MBP and PLP are localized, than of associated oli-godendroglial membranes, in which MAG and CNP are concentrated. Similar results have been obtained in several dysmyelinating mutants, pointing to common factors in virally and genetically caused hypomyelination. Key Words: Border disease—Myelin—Hypomyelination—Development—Sheeo.     

In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: Immunoblot identification of acylated PLP and DM-20

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.     

Rapid metabolism of fatty acids covalently bound to myelin proteolipid protein

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains approximately 2 mol of covalently bound fatty acids. In this study, the in vivo turnover rate of the acyl chains bound to PLP was determined in 40-day-old rats after a single intracranial injection of [3H]palmitic acid. The apparent half-life of total fatty acids bound to PLP was approximately 7 days. After correction for acyl chain interconversion, the half-life of palmitate bound to PLP was only 3 days. This turnover rate is much more rapid than that of the protein moiety calculated under the same experimental conditions (t1/2 = 1 month). Additional evidence for the dynamic metabolism of acyl groups was provided by experiments in brain tissue slices which showed that acylation of PLP occurs in adult animals as well as during active myelination. Acylation of endogenous PLP in purified myelin and its subfractions was also studied during rat brain development using either [3H]palmitoyl-CoA or [3H]palmitic acid plus ATP and CoA. Labeling of endogenous PLP with [3H]palmitoyl-CoA was observed as early as 10 days postnatal and continued at the same rate throughout development. When [3H]palmitic acid was used as precursor in the presence of both ATP and CoA, esterification of myelin PLP occurred rapidly in adult animals, indicating that both nonacylated PLP and acyl-CoA ligase are present in myelin. Finally, pulse-chase experiments in a cell-free system showed that PLP-bound fatty acids turn over with a half-life shorter than 10 min. These observations are consistent with the concept that acylation of myelin PLP is a dynamic process involved mainly in myelin maintenance and function.     

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.     

Adrenalectomy of Rats Results in Hypomyelination of the Central Nervous System

The effect of adrenalectomy on CNS myelin accumulation was investigated to determine whether glucocorticoids play a role in regulating myelination. When 14-day-old rats were adrenalectomized and sacrificed 7-8 days later, the amount of bulk-isolated myelin in whole brain, as expressed per gram wet weight of brain or per milligram DNA-phosphate, was reduced to about 75% that of sham-operated controls. Both brain weight and DNA content were unchanged by adrenalectomy. Examination of individual brain regions also revealed decreased amounts of myelin in adrenalectomized animals. Brain glycerol 3-phosphate dehydrogenase specific activity was reduced in adrenalectomized animals to 40-60% that of controls, and serum corticosterone levels were less than 0.6% of control levels. The amount of cerebral myelin in animals adrenalectomized on day 21 and sacrificed 9 days later was not significantly reduced. This suggests a possible role of glucocorticoids during the early period of rapid myelination.     

Maternal deficiency of protein or protein and calories during lactation: effect upon CNS myelin subfraction formation in rat offspring.

The present study has examined the effects of maternal protein and protein-calorie deficiency during lactation on the development of CNS myelin subfractions in rat offspring. The offspring of both the protein and protein-calorie deficient rats had decreased brain and body weights, as well as delayed CNS myelination. Delayed active CNS myelination was demonstrated by the fact that 53-day-old nutritionally stressed pups incorporated significantly more [³H]leucine and [¹⁴C]glucose into all myelin subfractions than age-matched controls. Delayed myelination was also supported by the tremendous accretion of myelin proteins in the nutritionally deprived pups between 25 and 53 days of age. Despite the delayed active synthesis of myelin, the myelin deficit persisted in the offspring of protein deficient rats. These offspring had a deficiency of light + medium myelin throughout the study. At 17 days, both groups of nutritionally stressed rats had an excess of the high molecular weight proteins in heavy myelin. Heavy myelin from 17 day offspring of protein-calorie deficient rats had a deficiency of basic proteins, while that from the offspring of protein deficient rats had a deficiency of proteolipid protein. The protein composition of all myelin subfractions was normal at 53 days.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated Myelinogenesis by Dietary Lipids in Rat Brain

Abstract: Our previous work showed an early development of behavioral reflexes in rats whose mothers had been fed, during pregnancy and lactation, a lipid fraction extracted from yeast grown on n -alkanes (which contain 50% odd-chain fatty acids) in comparison with controls fed a margarine diet. To clarify whether the observed changes might be linked to an early myelination, we have investigated mRNAs involved in myelin synthesis in the brains of offspring at 5 days of age by northern blot and in situ hybridization. Northern blot analysis showed that proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) mRNAs were higher in animals on the lipid diet compared with controls. In situ hybridization with probes specific for PLP, myelin basic protein, and MOG mRNA showed significantly higher numbers of positive cells in test animals compared with controls in all brain regions. This study shows an acceleration of myelinogenesis induced by dietary lipids. These data can give a new insight in the therapeutical approaches involved to promote repair in demyelinating diseases.     

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.     

THE EFFECTS OF UNDERNUTRITION ON THE PROTEINS OF OPTIC AND SCIATIC NERVES DURING DEVELOPMENT

Abstract— Polyacrylamide gel electrophoresis has been used to assess the appearance of some optic and sciatic nerve proteins in normal developing rats and in undernourished rats. Of the myelin proteins, the'Wolfgram'proteolipid is already present about the time myelination begins. The basic myelin proteins appear later, first in sciatic and then in optic nerve. A non-myelin basic protein, assumed to be a histone, is present at high levels in both nerves before myelination begins. There is no apparent effect of undernutrition on the appearance and amount of myelin proteins at 12, 16 and 22 days of age. The'histone'protein is reduced in optic and sciatic nerves at times corresponding roughly to the transition periods from cellular proliferation to myelin formation. The possibilities are discussed that myelin basic proteins are synthesized as compact myelin formation occurs, and that there may be retarded cellular proliferation in nerves of undernourished rats.     

ASTROGLIAL UPTAKE IS MODULATED BY EXTRACELLULAR K+

Abstract— Developmental changes of myelin proteins in chick sciatic nerve were studied at the stage of myelination by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The myelin of adult hen peripheral nervous system (PNS) contained two glycoproteins (BR-P0 and PASII), both of which are unique to PNS myelin, in addition to the basic encephalitogenic protein, BP, which is common to CNS and PNS myelin. The other basic protein (BF-P2) found in the PNS of other species was not definitely detectable in hen PNS. At the early stages of myelination (from 14 to 18 embryonic days) the amounts of myelin proteins increased rapidly in parallel with the increase in number of layers of the myelin sheath of the PNS. At 14 embryonic days high molecular weight proteins were dominant, while myelin specific proteins were barely detectable in the PNS myelin fraction. At 18 embryonic days, however, BR-PO, BP and PASII proteins became the main protein components of the PNS myelin, whereas high molecular weight proteins decreased in quantitative importance during development. At the early stage of myelination other glycoproteins were also detectable in the PNS myelin. Radioactive fucose was actively incorporated into the two glycoproteins, BR-P0 and PASII, at the early stage of myelination in vivo. These results suggested that myelin proteins especially glycoproteins, may play an important role in PNS myelin formation.     

Recovery of Proteolipid Protein in Mice Heterozygous for the Jimpy Gene

We have measured levels and synthesis of proteolipid protein (PLP) and its transport into myelin in female mice heterozygous for the jimpy gene and in their normal female littermates. In both cord and cerebrum, jimpy carriers show deficits in PLP during development followed by compensation in adulthood. Recovery of PLP occurs earlier in cord than in brain. At 13 days levels of PLP in carriers compared to controls are reduced to 0.60 and 0.44, respectively, in cord and cerebrum. By 100 days, normal levels of PLP are attained in cord (1.13) whereas levels of PLP in cerebrum are only 0.78 of control. By 200 days full recovery occurs in cerebrum, with a ratio of 1.21, suggesting a possible over-compensation. The yield of myelin from cerebrum was reduced to 0.78 in carriers compared to controls at 17 days. In brain slices, incorporation of [3H]leucine into homogenate PLP from carriers is the same as in controls, whereas [3H]leucine incorporation into myelin PLP is reduced to 0.68 of control. These results indicate that synthesis of PLP in the carriers is normal at 17 days, but transport of PLP into myelin is reduced. Similarly, acylation of homogenate PLP is normal, whereas acylation of myelin PLP is reduced, as measured by incorporation of [3H]palmitic acid. Transport of PLP into myelin was compared to transport of MBP; transport of both proteins was equally decreased as indicated by the similar ratio of labeled PLP to MBP in myelin from carriers compared to noncarriers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Emergence of three myelin proteins in oligodendrocytes cultured without neurons

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin-associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.     

Myelin-Associated Glycoprotein and Other Proteins in Trembler Mice

The myelin-associated glycoprotein (MAG) and other myelin proteins were quantitated in homogenates of whole sciatic nerve from adult and 20-day-old Trember mice. In the nerves of adult mice, the concentration of MAG was increased from 1.1 ng/micrograms of total protein in the controls to 1.4 ng/micrograms protein in the Tremblers. By contrast, the concentrations of P0 glycoprotein and myelin basic proteins were reduced to 27% and 20% of control levels, respectively. Immunoblots demonstrated that P2 was also greatly reduced in the Trembler nerves. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was 65% of the control level. Immunoblot analysis showed that MAG had a higher than normal apparent Mr in the sciatic nerves of the Trembler mice, but its apparent Mr was normal in the brains of these mutants. In 20-day-old Tremblers, the P0 and myelin basic protein were reduced slightly less to about 40% of the level in the nerves of age-matched controls. CNP and MAG levels were not significantly different from those in controls, and MAG exhibited a shift toward higher apparent Mr similar to that in the adults. The maintenance of high MAG levels despite the severe deficit of myelin, as reflected by the decrease of the major myelin proteins, is consistent with the immunocytochemical localization of MAG in periaxonal Schwann cell membranes, Schmidt-Lantermann incisures, lateral loops, and the outer mesaxon and its absence from compact myelin. The abnormal form of MAG in the peripheral nervous system (PNS) of the Trembler mice may contribute to the pathology in this mutant.