Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.     

Immunoblot Identification of 13.5 Kilodalton Myelin Basic Protein in Goldfish Brain Myelin

Myelin isolated from goldfish brain shows a multilamellar structure with a major dense line and two intraperiod lines. Sodium dodecyl sulfate gel electrophoresis revealed that the protein profile of goldfish brain myelin is distinctly different from that of rat brain myelin. No protein migrating to the position of proteolipid protein or DM-20 was seen in goldfish myelin. Goldfish acclimated to 5 degrees, 15 degrees, and 30 degrees C showed no qualitative differences in myelin proteins. The 13.5 kD protein in goldfish brain myelin and brain homogenate was intensely immunostained with the antiserum to human basic protein by the immunoblot technique. In contrast, none of the proteins of goldfish myelin were immunostained with antiproteolipid protein serum; however, both proteolipid protein and DM-20 of rat brain myelin were immunostained. The significance of the synthesis of myelin proteins by astrocytes in the goldfish brain is discussed.     

Alterations in mRNA Expression of Myelin Proteins in the Sciatic Nerves and Brains of Streptozotocin-induced Diabetic Rats

Diabetic neuropathy is the most common complication of diabetes. We examined the levels and the mRNA expression of myelin proteins in the sciatic nerves and the brains of streptozotocin-induced diabetic rats. The diabetic rats exhibited a decrease in body weight, elevation of the blood glucose level and a decrease in motor nerve conduction velocity at 2 weeks after streptozotocin injection. In the sciatic nerves of diabetic rats, the level of P0 protein and its mRNA expression were markedly reduced at 20 weeks after the injection. In the brains, the levels of proteolipid protein and myelin-associated glycoprotein and their mRNA expression were selectively decreased at 20 weeks after the injection. This affected expression of myelin proteins was found even when no histological abnormalities were detectable. Considering the functional significance of myelin proteins, this impairment of protein expression is possibly involved in the pathogenesis of diabetic neuropathy, including that in brain disorders.     

Uridine Transport and Metabolism in the Central Nervous System

Myelin and myelin-containing (P3) fractions were prepared from human white matter by discontinuous sucrose gradient centrifugation. The myelin isolated from each of the fractions of different densities was morphologically and biochemically distinct. Light myelin fractions consisted of compact, multilamellar myelin, whereas the denser fractions consisted predominantly of loose myelin with fewer lamellae. The amounts of both basic protein and lipophilin (proteolipid protein) were reduced in the denser fractions. In contrast, the high-molecular-weight components were elevated in the dense fractions. The lipid composition was similar in all the fractions studied. Analysis of basic protein by gel electrophoresis at pH 10.6 revealed differences in basic protein microheterogeneity among the fractions. The light myelin fraction was enriched in the more positively charged basic protein components (components 1, 2, and 3), whereas these components were reduced in the denser fractions. Myelin in the dense fractions was enriched in the more modified forms of basic protein (components 6, 7, and 8). The pattern of microheterogeneity was different for basic protein isolated from myelins of a 2-year-old and an adult brain; the former showed fewer components and mainly the most cationic species. On the other hand, the pattern of microheterogeneity of basic protein isolated from the different density gradient fractions was similar for both ages.     

Effects of Altered Thyroid States on Myelinogenesis

Abstract: Myelinogenesis was studied in controls and in rats treated since birth with Methimazole (hypothyroid) or thyroxine (hyperthyroid). The amount of myelin in forebrain and its protein composition were determined between 13 and 40 days of age, the period of most rapid myelin accumulation. Hypothyroid rats had reduced body and brain weights relative to controls and the yield of myelin was reduced on both a per brain and a per milligram brain protein basis. Developmental changes in the protein composition of isolated myelin followed the pattern of control animals (the percentage of total myelin protein present as proteolipid protein, large basic protein, and small basic protein increased, as did the ratio of proteolipid/large basic protein) but were delayed temporally by 1–2 days. Hyperthyroid rats also had reduced body and brain weights. At 13 days myelin accumulation was greater than that of controls, corresponding to an earlier initiation of myelination. At later ages myelin yield was reduced on a per brain basis but not on a per milligram brain protein basis. The developmental pattern of myelin protein composition was accelerated temporally by 1–2 days. Myelination in optic nerve, assayed by proteolipid protein content, also was slightly delayed in hypothyroid animals and somewhat accelerated in hyperthyroid animals. The relative synthesis of myelin proteins (determined as incorporation of intracranially injected [³H]glycine into myelin protein relative to incorporation into whole brain protein), as well as distribution of radioactivity among individual myelin proteins, was determined. The results supported the conclusion of the myelin protein accumulation study; hypothyroidism retards the developmental program for myelinogenesis, whereas in the hyperthyroid state myelin synthesis is initiated earlier but is also terminated earlier.     

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.     

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)     

Expression of Proteolipid Protein Gene Is Directly Associated with Secretion of a Factor Influencing Oligodendrocyte Development

Abstract: Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild-type cells in vivo and in vitro. It is possible that the presence of PLP or DM-20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM-20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM-20-producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM-20. By addition of the supernatants from the PLP/DM-20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.     

Demyelination Induced in Aggregating Brain Cell Cultures by a Monoclonal Antibody Against Myelin/Oligodendrocyte Glycoprotein

A monoclonal antibody (8-18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti-MOG antibody in the presence of complement, myelin basic protein (MBP) concentration decreased in a dose-related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase. In the absence of complement, anti-MOG antibody did not induce detectable demyelination. In contrast to the effect of anti-MOG antibody and as expected, anti-MBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side of the myelin membrane, is a good target antigen for antibody-induced demyelination. Indeed, they show that a purified anti-MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin-associated glycoprotein. These observations may have important implications regarding the possible role of anti-MOG antibodies in demyelinating diseases.     

Presence of the Plasma Membrane Proteolipid (Plasmolipin) in Myelin

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.