Notes: Abstract: Ganglioside synthesis and transport to myelin was studied in brainstem slices prepared from 19–21-day-old rats. The slices were incubated for up to 2 h in the presence of [3H]glucosamine to label primarily the hexosamine portion of complex gangliosides. The amount of radioactivity incorporated into gangliosides during slice incubations was only 10–15% of the amount of the label incorporated during in vivo labeling of brainstem gangliosides using equivalent amounts of [3H]glucosamine. Among individual gangliosides this inhibition was greater for the more complex gangliosides. When labeled gangliosides were isolated from homogenate and myelin fractions prepared from brain slices, the complex total gangliosides of both fractions showed a lag in labeling kinetics but with a lower specific radioactivity for the myelin fraction, reflecting the larger pool size and slower turnover rate exhibited by myelin components. Chase experiments showed that more complex gangliosides in homogenate exhibited almost no effect of chase after 30 min. Addition of the Golgi-disrupting agent monensin to slice incubations inhibited the labeling of all gangliosides except GM3, GM2, and GD3, and transport to myelin of all complex gangliosides except GM2. These results show that a monensin-sensitive mode of transport is responsible for the translocation of most newly synthesized gangliosides into myelin.

Notes: Abstract: 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 overcompensation. 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]Ieucine 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. Thus, total synthesis of PLP is not reduced in the carriers, whereas transport of both PLP and MBP into myelin is decreased, as are levels of myelin. Our data from the metabolic studies and measurement of PLP show that myelination is retarded in the carriers. Recovery from the deficit in myelin appears complete by later ages. We find, however, no evidence for a selective defect in synthesis or acylation of PLP, or transport of PLP relative to MBP. Mechanisms that could be employed by the oligodendrocyte to compensate for the early deficits in the carriers are discussed relative to glial differentiation and the possible formation of defective PLP.

Notes: Abstract: Brain and spinal cord of female mice heterozygous for the jimpy gene were analyzed during development for activity of ceramide galactosyl transferase (CGT) and for levels of myelin basic protein (MBP). CGT activity was low at 13–14 days in brains of heterozygous jimpy females but showed normal levels by 31 –36 days, in agreement with our earlier study of this enzyme. In cord, CGT activity was normal or slightly above normal at all ages studied, from 13–14 days into adulthood. In both brain and cord, decreased levels of MBP were observed at 13 days; by 100 days, amounts of MBP approached normal levels. Proven female carriers of the jimpy gene also showed normal levels of CGT activity, MBP, and isolated myelin at 200–250 days of age in both brain and cord. These biochemical findings agree with previous morphologic measurements in cord demonstrating deficits in myelin at early ages but compensation by 100 days. Our results show that compensation occurs earlier in cord than in brain and that levels of MBP show a closer correlation than CGT activity with amounts of myelin, as measured by either morphometric analysis or direct isolation.

Notes: Abstract: Monensin and colchicine have been used in a variety of systems to disrupt functioning of the Golgi apparatus and transport of Golgi-derived vesicles to the plasma membrane. In this study the effects of monensin and colchicine on the synthesis of cerebroside and sulfatide and their appearance in myelin were examined to determine whether these myelin components are processed through the Golgi apparatus. Brain slices from rats 17 days old were incubated with [3H]galactose and [35S]sulfate to label cerebroside and sulfatide. Myelin was isolated on sucrose density gradients. Fractions highly enriched in cerebroside and sulfatide were prepared from homogenates and myelin fractions by lipid extraction, alkaline methanolysis, and in some cases TLC. Monensin at 0.1 μM had no significant effect on synthesis of these galactolipids as measured by incorporation of [3H]galactose into cerebroside or [35S]sulfate into sulfatide in homogenates. However, appearance of [35S]sulfatide in the myelin fraction was reduced to 49% of control, while appearance of [3H]cerebroside was not significantly reduced. Colchicine from 1 mM to 0.1 μM had effects similar to monensin, that is, appearance of [35S]sulfatide in myelin was depressed, but again [3H]cerebroside was not affected. Incorporation of [35S]sulfate into sulfatide in homogenate was 93% of control, while appearance of [35S]sulfatide in the myelin fraction was depressed to 58% of control. The inhibition of appearance of sulfatide in myelin by colchicine and monensin is consistent with the view that sulfation of cerebroside occurs in the Golgi and that sulfatide is transported via Golgi-derived vesicles to the forming myelin membrane. Further, synthesis of cerebroside and its appearance in myelin are not inhibited by colchicine or monensin, indicating that cerebroside destined for myelin is not processed through the Golgi apparatus.

Notes: Abstract: Expression of the jimpy gene in heterozygous females was analyzed by measuring galactolipid synthesis in brain during early myelination. Sulfatide labeling in brains of heterozygous females at 13–14 days is decreased to 40–80% that of female littermates who do not carry the jimpy gene. The activities of ceramide galactosyl transferase and cerebroside sulfotransferase and levels of myelin basic protein were similarly depressed. Since the jimpy gene was maintained with the Tabby gene in these studies, the effect of the Tabby gene on these parameters was examined and found to have no effect. These biochemical findings indicate that myelination is retarded in the brains of heterozygous jimpy females during the second week of development. However, at 18 days and thereafter, sulfatide labeling is less reduced, suggesting that the oligodendrocytes in brain attempt to compensate, in agreement with morphologic studies which show that myelin is decreased in brain during early development, but appears normal in adult animals.

Notes: Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [3H]fucose or [14C]glycine precursors. The nerve slice system gave nearly linear incorporation of [3H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [3H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10−8m to 1.5 × 10−6m. Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P0, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P0 into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P0 myelin.

Notes: Brain slices were prepared from 17-day old rats, and incubated with [3H]glycine or [3H]-leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one-fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min.When cycloheximide (0.2 mM) or puromycin (1.0 mM) was added, appearance of newly labeled basic and Wolfgram proteins in myelin stopped while proteolipid protein continued to appear in myelin at a normal rate for at least 30 min. Chase experiments with unlabeled glycine had similar effects. These results indicate the existence of a previously synthesized precursor pool of proteolipid protein with a 30-min interval between synthesis of proteolipid protein and its appearance in myelin.Incorporation of [3H]fucose into glycoprotein of the myelin sheath was studied, as was inhibition of incorporation of radioactivity by the use of either cycloheximide, or dilution with unlabeled fucose. The results indicated fucosylation of a sizable pool of presynthesized protein and a delay of 30 min between fucosylation of these polypeptides and their subsequent appearance in myelin as glycoproteins.

Notes: Abstract— Partially purified myelin from brains of 17-day-old rats was separated into 4 subfractions on a discontinuous sucrose gradient by virtue of heterogeneity in density and particle size. The protein composition of each subfraction was determined by densitometry following separation of proteins on polyacrylamide gels in buffers containing sodium dodecyl sulphate. The major proteins studied included two basic proteins, proteolipid protein, the major high molecular weight protein (W) and a group of high molecular weight proteins.The percentage of high molecular weight proteins decreased sequentially from fraction D to A, that of the W protein remained constant, while relative amounts of the two basic proteins increased. Proteolipid protein concentration also increased as a percentage of the total protein from fraction D to B, but the uppermost fraction. A, had a markedly lower amount than fraction B. At 1 h after intracranial injection of [3H]leucine, the specific radioactivity of the basic and proteolipid proteins decreased from fraction D to B, with proteolipid protein in fraction A again anomalous (specific radioactivity higher than expected). These results are consistent with (but do not prove) a precursor-product relationship for individual proteins from denser to lighter subfractions, with the exception of myelin subfraction A.Experiments involving time staggered injections of a [14C] and later a [3H] labelled amino acid gave data which demonstrated that the W and basic proteins were added simultaneously (or with delays of much less than 20 min) to all of the subfractions, while proteolipid protein was added sequentially, from lower to upper fractions on the gradient. This double isotope technique also confirmed our previous observations that proteolipid protein shows a lag in entry into myelin compared to basic protein.

Notes: Abstract— When [2-3H]glycerol was injected intracranially into young rats, it was presented as a pulse label, leaving the brain rapidly and giving up much of its labelled hydrogen to water. [2-3H]glycerol was efficiently incorporated into brain lipids, especially into choline and ethanolamine phospholipids. Following injection of a mixture of [3H]- and [14C]-labelled glycerol, the ratio of 3H to 14C in the phospholipids of both whole brain and the microsomal fraction decreased as a function of time after injection. This finding indicated less recycling of the tritium label. This lack of recycling was further indicated by the finding that 94 per cent of the tritium label of phosphatidyl choline was in the glycerol portion of the molecule rather than in the fatty acids. At 2 weeks following injection with [3H]glycerol, 93 per cent of the total radioactivity in brain appeared in the lipid fraction. In contrast, following injection with [14C]glycerol, only 57 per cent of the radioactivity appeared in lipid, with about 20 per cent in protein.

Notes: Abstract— Brain slices from 17 day rats were incubated with [3H]galactose and [35S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of [3H]cerebroside and [3H]sulphatide in myelin showed a lag of less than 15min, while appearance of [35S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of [3H]cerebroside and [3SS]sulphatide in the non-myelin fraction and of [3H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, [35S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while 3H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non-myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non-myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non-myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with [2-3H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra-myelin site, or synthesis of the lipids within myelin itself.