Early Stages Of Myelination Research Articles

Analysis of integrin expression on oligodendrocytes during axo-glial interaction by using rat-mouse xenocultures

To analyze the expression of cell-surface molecules on neurons or glia during myelination, we have developed a xenotypic coculture system in which mouse oligodendrocytes interact with rat dorsal root ganglion neurons. The axo-glial interactions in these cultures promote oligodendrocyte precursor cell proliferation, survival, and differentiation as in vivo, thus supporting the validity of the xenocultures as a model system to study myelination in which the molecules on the neurons or the glia can be distinguished using species-specific antibodies. We examined the expression of integrins, the major family of cell-surface extracellular matrix receptors, on oligodendrocytes during the early stages of myelination and found that, unlike Schwann cells, oligodendrocytes do not express alpha 6 beta 4 in association with myelin sheath formation. The pattern of integrin expression observed on oligodendrocytes in these cultures is similar to that seen in oligodendrocytes that differentiate in purified cultures, and it comprises alpha 6 beta 1, alpha v beta 5, and an as yet uncharacterized alpha v-associated beta subunit of 80 kDa. Changes in integrin expression associated with differentiation, therefore, do not depend on axonal contact, and beta 4 is not required for myelin sheath formation, although its expression may contribute to the individual properties of oligodendrocytes and Schwann cells.

Expression of the Myelin Basic Protein Gene in Transgenic Mice Expressing Human Neurotropic Virus, JCV, Early Protein

Transgenic mice containing the early region of the JC virus encoding T-antigen developed neurological disease resulting from dysmyelination in the central nervous system. In this study, we investigate expression of the myelin basic protein (MBP) gene, a major constituent of the myelin sheath, at the RNA level by Northern blot and S1 nuclease assay and at the protein level by Western blot analysis using anti-MBP antibody in two distinct transgenic lines exhibiting different degrees of dysmyelination. Results from Western blot analysis of proteins from the brains of these mice revealed great reductions in MBP levels that parallel the severity of dysmyelination in the corresponding animals, Analysis of MBP RNA by Northern and quantitative S1 assays exhibited no alterations in the transcription initiation sites of the MBP gene in these animals; however, a significant decrease in the level of MBP mRNA was detected, suggesting that T-antigen may negatively influence transcription of the MBP gene. Results from Northern and Western blot analysis of proteolipid protein revealed low-level expression of this gene. Expression of JCV T-antigen is developmentally regulated in the transgenic mice; it appears at 8′ days postnatal, peaks at 15 days, and substantially decreases in 18-day-old mice. The programmed expression of JCV T-antigen, which overlaps With MBP gene transcription at the early stage of myelination, suggests the involvement of a pathway which modulates stage-specific regulation of myelin genes and viral gene expression in transgenic mice during brain development.

Clustering and mobility of voltage-dependent sodium channels during myelination

In myelinated axons, voltage-dependent sodium channels are segregated at high density at nodes of Ranvier (Rosenbluth, 1976; Waxman and Quick, 1978; Black et al., 1990; Elmer et al., 1990), a distribution that is critical for the saltatory conduction of action potentials (Huxley and Stampfli, 1949). The factors that specifically control the organization and immobilization of sodium channels at nodes are unknown. Recently we have reported that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to maintain sodium channel distribution during differentiation of myelinated nerve (Joe and Angelides, 1992). The specific recruitment of sodium channels at these early stages of myelination and the conspicuous absence of other axon membrane components suggest that the factors governing sodium channel cluster formation show molecular specificity. However, it is not clear whether these clustered sodium channels originate from a redistribution of preexisting diffusely distributed sodium channels. To determine how Schwann cells might regulate sodium channel distribution during myelination we have examined the lateral mobility of fluorescently labeled sodium channels at defined stages of myelination by fluorescence photobleach recovery using tetramethylrhodamine (TmRhd)-labeled Tityus gamma, a sodium channel-specific fluorescent toxin. First, to test whether Schwann cells, in addition to modulating sodium channel distribution, affect the mobility of sodium channels, we cultured dorsal root ganglion neurons in the presence or absence of Schwann cells and monitored sodium channel mobility on cell bodies, axon hillocks, and axons. Even in the absence of Schwann cells, approximately 80% of the sodium channels were immobile on the time scale of the fluorescence photobleach recovery measurement (DL < or equal to 10(-12) cm2/sec), although the remaining fraction of channels are mobile with diffusion coefficients of 5-13 x 10(-11) cm2/sec. Most importantly, in contrast to the effects of Schwann cells on altering the distribution of sodium channels, we found that Schwann cells did not alter the rate of lateral mobility or the mobile fraction of axonal sodium channels. Therefore, although sodium channel distribution depends on Schwann cell contact, immobilization of sodium channels is independent of Schwann cell contact. These effects appear to be specific for sodium channels because 45% of the succinyl concanavalin-A receptors on the axon are mobile, a fraction that decreases to 25% in the presence of Schwann cells later in development. To determine how sodium channels might be immobilized other than by Schwann cell contact, TmRhd-Tityus gamma-labeled dorsal root neurons were treated with 0.5% Triton X-100.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental Expression of Myelin Protein Genes in Dysmyelinating Mutant Mice: Analysis by Nuclear Run‐Off Transcription Assay, In Situ Hybridization, and Immunohistochemistry

Gene expression for myelin proteolipid protein (PLP) and myelin basic protein (MBP) in the dysmyelinating mutant mice shiverer and jimpy was analyzed by nuclear run-off transcription assay, in situ hybridization, and immunohistochemistry. The level of PLP transcription in shiverer brains was lower than that in controls at postnatal day 18 but relatively higher at later stages. In spite of the considerable amount of hybridization with PLP cDNA, immunoreaction for PLP was greatly reduced in shiverer mice throughout their lives, probably owing to a defect in the assembly of PLP into myelin. Abnormal deposition of PLP in oligodendroglial cell bodies suggested that transport of PLP to myelin is delayed in shiverer brains. The number of oligodendrocytes expressing PLP mRNA was drastically reduced in jimpy mice. MBP mRNA in jimpy mice is localized preferentially in oligodendroglial cell bodies, a result suggesting that oligodendrocytes in jimpy are mostly the immature type. Although transcriptional activity of the MBP gene in jimpy was greatly reduced, a finding reflecting the decrease in the number of mature oligodendrocytes, that of the PLP gene remained high at early stages. The discrepancy of the two gene expressions is discussed relative to the role of PLP transcripts at early stages of myelination.

Identification of GTP‐ binding proteins in myelin and oligodendrocyte membranes

Myelin membranes purified from mouse and rat brain are associated with alpha subunits of four signal transducing guanosine triphosphate (GTP)-binding proteins: Go, Gi, Gs, and ras. Four low-molecular-weight (Mr) GTP-binding proteins are also present, as demonstrated by the binding of GTP to proteins immobilized in nitrocellulose. This latter group is more prominent at early stages of myelination and remains associated with isolated myelin membranes despite repetitive cycles of purification. At least one nonmyelin subcellular membrane fraction possesses the same proteins. The total membrane fraction of cultured oligodendrocytes is associated with both groups of GTP-binding proteins. None of the well-known myelin proteins bound GTP by the procedure described.

Enzymes that degrade phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate have different developmental profiles in chick brain

The activities and subcellular distributions of the hydrolases that degrade polyphosphoinositides were compared in the developing chick central nervous system. Specific activities increased 2- 3-fold and total activities increased 13- to 16-fold. Phosphatidylinositol 4-phosphate phosphatase is localized in membranes (78%), but is preferentially associated with nonmyelin membranes, since the increase in specific activity preceded myelination and proportions of membrane and soluble activities were constant during accumulation of myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphatase is largely soluble in embryonic (57%) and myelinated brain (50%). Although specific activity increased coincident with myelination, approximately equal increases in soluble and membrane activity indicate no preferential association with myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphodiesterase activity increased only in the early stages of myelination, but showed some preferential association with myelin membranes, since the proportion of soluble diesterase declined from 40 to 25%.

Two Isoforms of Myelin-Associated Glycoprotein Accumulate in &lt;i&gt;Quaking &lt;/i&gt;Mice: Only the Large Polypeptide Is Phosphorylated

We have shown that the developmentally regulated appearance of the two myelin-associated glycoprotein (MAG) polypeptides in normal mouse brain myelin does not reflect the developmental pattern of differential splicing of primary gene transcripts as determined earlier by RNase protection assays. Contrary to expectation, the large (L-MAG) and small (S-MAG) polypeptides are present in about equal amounts at a relatively early stage of myelination, day 24 or earlier. In quaking (qk) mutant mouse brain myelin, both MAG polypeptides are evident at all ages examined; the relatively greater abundance of S-MAG compared to L-MAG at early ages (days 18 and 22) confirms our earlier observation on in vitro translations of mRNA. At later ages (day 27 and beyond) both isoform are present in approximately equal amounts. The L-MAG but not the S-MAG polypeptide can be phosphorylated by kinases that are endogenous to isolated qk myelin, analogous to the phosphorylation we have observed in vivo in normal mice and in their isolated myelin.

Temporary adhesions between axons and myelin-forming processes

Following irradiation, the dorsal funiculus of the lumbosacral spinal cord in the rat undergoes the following sequence of events: (a) a marked reduction of the normal glial population, (b) an absence of oligodendrocyte myelin formation, (c) the invasion and proliferation of Schwann cells, and (d) the myelination of axons within the cord by Schwann cells. The present study demonstrates that, during the latter process, junctional complexes develop between these intraspinal Schwann cells and the axolemma. These complexes are present at sites of probable initial contact between the two membranes. As the Schwann cell process begins to wrap the axons, these junctional complexes are located between the inner spiraling process of the Schwann cell and the axon. With the advancement of myelin formation to the stage of 8 to 9 compact spirals, these contacts are rarely observed. Spinal cords from normal 8-day-old rats were examined in order to determine if such contacts occur during myelination by oligodendrocytes. Although they are more difficult to detect in the normal animal due to the abundance of glial processes, similar junctional complexes occur between oligodendrocyte processes and axons. These observations suggest that these complexes may serve to stabilize and to guide the myelin-forming process around the perimeter of the axon. Additionally, these junctions may play an active role in the advancement of the inner spiraling process by forming temporary adhesions between the axolemma and the adjacent myelin-forming process. Coated vesicles are commonly observed fused with the axolemma of axons which are in the early stages of myelination. These coated vesicles may be involved in the insertion or the deletion of junctional membrane.

Development of axonal-oligodendroglial relationships and junctions during myelination of the optic nerve

The early stages of myelination were examined in optic nerves of rats aged 12–15 days. The initial association between oligodendroglial processes and bare axons involves no junctional specialization, as the axoglial extracellular space remains unaltered. Following ensheathment by a collar of glial cytoplasm, at least one full rotation of mesaxon was evident before compact myelin formed. Furthermore, myelin was generally evident before a second rotation was completed. In longitudinal sections, an axoglial junction was always observed beginning on the first paranodal loop, continuing through to the last (or outermost) loop. Thus, the formation of myelin and elaboration of a junctional complex in the paranodal region follow a promyelination phase and appear to be synchronous (and possibly related) events. Although the paranodal plasmalemma and axolemma are in close apposition, there is a material in the extracellular space that precipitates phosphotungstic acid, a characteristic that appears to be featured in a number of different types of cell junctions.

P2, P1, and P0 myelin protein expression in developing rat sixth nerve: A quantitative immunocytochemical study

Myelination and the expression of myelin proteins P2, P1, and P0 were studied quantitatively in the rat sixth cranial nerve during development. The postnatal development and growth of all myelin sheaths in this nerve have been studied morphometrically in a companion paper. Epon-embedded blocks with closely matched topography in the transverse plane were selected from rats perfused at ages 1-4, 8, 15, and 20 days. From each block, serial semithin sections were cut, etched, and immunostained according to the peroxidase-antiperoxidase method with well-characterized polyclonal antisera that reacted specifically with P0 glycoprotein and the basic proteins P1 and P2. The immunoreactivities of individual myelin sheaths were measured by densitometry. Numbers of compact myelin lamellae, myelin spiral lengths, and axon diameters were determined on electronmicrographs of adjacent thin sections. At birth anti-P0 immunoreactivity was found on sheaths with two and more compact lamellae; neither P1 nor P2 immunoreactivity was observed. On day 2, myelin sheaths with five and eight lamellae were stained respectively by anti-P1 and anti-P2. On day 3 the percentages of myelin sheaths stained were substantially higher: P0 95%, P1 78%, P2 15%. By day 4, anti-P0 and anti-P1 immunoreactivity was present in 95% of myelin sheaths; 35% were stained by anti-P2. For P2, staining intensity and percentage of myelin sheaths stained continued to increase and by day 20, 85% were anti-P2-positive. The density of immunoreactivity was not uniform in all myelin sheaths. At young ages staining varied with all three proteins. The variability decreased as myelin sheaths thickened; it persisted longest for anti-P2. We conclude that the density and distribution of immunoreactivities of P0, P1, and P2 reflect their relative concentrations during myelin sheath development and growth. We attribute lack of detectable anti-P2 immunoreactivity in some small sheaths at 20 days to their early stage of myelination and also to limitations of the method. We infer from our observations that all myelin-forming Schwann cells express P2 basic protein.

Developmental changes of myelin-associated glycoprotein in rat brain: study on experimental hyperphenylalaninemia

We examined developmental changes of myelin-associated glycoprotein (MAG), basic protein (BP), and proteolipid protein (PLP) in central nervous system myelin isolated from experimental hyperphenylalaninemic rats (PKU rats) and controls. Higher amounts of MAG, including high-molecular-weight MAG in myelin, were found in 12- to 21-day-old control rats than in adult rats. MAG in developing myelin was at a maximum in 18-day-old rats and began to decrease in 21-day-old rats, while PLP and BP in developing myelin increased at these developmental stages. The level of high-molecular-weight MAG decreased in myelin prepared from 21-day-old rats. These results suggest that the decreasing high-molecular-weight MAG is important for compaction of myelin in the early stage of myelination. In myelin from 12- to 18-day-old PKU rats, the ratio of each protein such as MAG, PLP, or BP to that of control was about 0.5 at 12 days, and increased to almost 1.0 at 18 days. The myelination seems to be initially delayed but to be close to that of controls in PKU rats about 18 days old.

Peripheral nerve fibres regenerate through myenteric plexus

The ability of myenteric glia and neurons to support peripheral nerve regeneration was tested by grafting pieces of muscularis externa 5 mm long from the distal colon of inbred CBA mice adjacent to the proximal stump of cut common peroneal nerves. By two weeks after operation many axons had invaded the plexus and after 3 weeks regeneration common peroneal nerve fibres could be identified in all parts of the plexus throughout the grafts. Some axonal profiles within the plexus appeared to be in the early stages of myelination by enteric glia. Axons surrounded by compact myelin were found at the periphery of ganglia, but the cells involved resembled Schwann cells and could not be positively identified as enteric glia. Profiles similar to those of regenerating axons were only very rarely seen in control experiments in which grafts were placed adjacent to intact common peroneal nerves. It is suggested that the cellular elements of the myenteric plexus can support peripheral nerve regeneration.

Dysmyelinogenesis in caprine β-mannosidosis: Ultrastructural and morphometric studies in fetal optic nerve

The optic nerves from a goat fetus affected with β-mannosidosis and a control fetus were analysed morphologically in order to investigate developmental aspects of β-mannosidosis-associated myelin deficits. In the affected fetus, the number of myelinated axons per unit area was about 25% of the control values. Histograms of axonal diameter indicated that a greater percentage of the myelinated and unmyelinated axons were of larger caliber in the affected fetus than in the control fetus and that very few small axons were myelinated in the affected animal. The mean values of myelin sheath thickness in the affected and control animals did not differ significantly. Ultrastructural analysis revealed a decreased proportion of oligodendrocytes and an increased proportion of astrocytes in the affected fetus.These results indicate that the pathogenetic process leading to cellular abnormalities and myelin deficits in β-mannosidosis has been initiated prior to 124 days gestation, during an early stage of myelination in the goat optic nerve. The decrease in number of oligodendrocytes suggests that early cell death and/or change in oligodendrocyte proliferation contribute to the myelin deficit. Analysis of the prenatal development of lesions will help clarify the pathogenesis of dysmyelinogenesis in β-mannosidosis.

Changes in monogalactosyl diacylglycerols, alkylgalactolipids and cerebroside fatty acid esters in maturing rat brain measured by high-performance liquid chromatography

A newly developed high-performance liquid chromatography method was used to measure four nonpolar glycolipids in developing rat brain. The accumulation patterns of nonhydroxy- and hydroxycerebroside fatty acid esters were similar to those of myelin cerebrosides. In contrast, both monogalactosyl diacylglycerol and alkylgalactolipids reached maximal levels at the early stages of myelination.

Defective differentiation of peripheral nerves in the dystrophic mouse.

Sections or teased nerve fibers from developing common peroneal (CPN), tibial (TN) and sural nerves (SN) of dy/dy and normal mice were morphometrically examined at light and electron microscope levels. The data show that: (1) in the dy/dy CPN and TN, and to a lesser degree in the dy/dy, SN onset of myelination is delayed compared to normal nerves during the first postnatal week. This correlates with a retarded progression of segregated axon → promyelin axon → myelinated axon, and with a reduced rate of growth of axons in diameter; (2) at the completion of myelination fewer than normal numbers of myelinated axons are produced in the dy/dy CPN and TN, while in the dy/dy SN the numbers are normal; (3) in the dy/dy CPN and TN, and less in the dy/dy SN average internode is shorter than normal from early stages of myelination. It elongates at a slower than normal rate but roughly in parallel with the dy/dy limb, which has normal length and rate of elongation; (4) in the dy/dy nerves shorter than normal average internode is attributable partly to delays in onset of myelination, partly to shorter than normal axonal territory acquired by Schwann cells at the onset, and, particularly in the dy/dy CPN and TN, partly to fewer than normal short internodes eliminated during maturation. We conclude that: (1) in the dy/dy peripheral nerves (PNS) abnormalities of myelination are established during the first 2 weeks of postnatal development; and (2) differentiation of the dy/dy motor PNS is more defective than that of the dy/dy sensory cutaneous PNS.

Gangliosides and proteins in developing chicken brain myelin.

Ganglioside and protein content of chicken brain myelin have been studied as a function of development and maturation. Ganglioside concentration remained relatively constant at approximately 225-266 micrograms sialic acid per 100 mg myelin, from 17-day-old embryos to 540-day-old adults. The ganglioside distributional pattern was also constant, GM1 and GM4 each accounting for approximately one-third of total sialic acid. These properties are contrasted with those of mouse and rat brain myelin which previously showed enrichment of GM1 at early stages of myelination and appearance of GM4 at a later stage of maturation. In an earlier study of the mouse total myelin gangliosides doubled in concentration between 3 and 5 weeks of age and full maturity. Myelin proteins of the chicken also remained relatively constant in concentration and distributional pattern during development, although the percentage of basic protein increased somewhat during the first few weeks after hatching. Correlations were observed between molar concentrations of basic protein and various gangliosides, particularly the monosialo types.

Myelin sheath maturation of intrapulmonary axon bundles in the rabbit fetus.

The development of intrapulmonary myelin sheaths began in the central parts of the lung and proceeded towards the periphery. It started suddenly on the 25th post conceptional (p.c.) day. Early stages of myelination could be demonstrated in the periphery on the 27th day p.c (2) Axons not surrounded by Schwann cells could be seen in large numbers on the 24th and 25th day p.c. but they became fewer on the following days. (3) Myelinated axons was found were often at the periphery of the axon bundle. The maximum number of myelin layers was 7 on the 25th day p.c. and 30 on the 31st day p.c. (4) Axons at early stages of myelination were seen along with axons with more progressive myelin sheath development. Abnormal structures of myelinated fibers could be demonstrated. Their significance is discussed.

Differentiation of cells in aggregates from human, mouse, and chick embryo brain. An electronmicroscopic study

Summary 1. The pattern of ultrastructural organisation of cell aggregates from 18-week-old human, 18 day-old mouse and 8-day-old chick embryo brains were studied with the help of EM, SEM, and freeze-etching techniques. 2. Until the 4–5 DIV the aggregates are composed of single microaggregates, which consist of groups of immature neurons. However during the first days in culture the formation of new processes from nerve cells starts. Newly formed processes invade space not occupied by cellperikarya-The later component of the neuropil are processes of glial cells, mostly of astrocytic origin. The neuropile of differentiated aggregates has a similar structure and the extracellular space is limited to the same extent as in vitro . 3. The organization of the surface of aggregates suggests, that degenerated cells are sequestered into the medium and migrate in or out of aggregates. Already during the 1st week, glycogen particles appear in aggregates and later glycogen accumulates to a remarkable extent in astrocytic processes and in dendrites. 4. Early stages of myelination and the precursors of synapses were found at the beginning of the 2nd week. Up to 4 till 5 week of cultivation signs of cell degeneration are evident, the neuropile thins and glia represents the majority cell population. 5. Freeze-etching supported the EM observations. The ultrastructural organisation of intracellular and cellular membrane complexes is well apparent. Pore complexes were observed in nuclear membranes. Their appearance and distribution is similar to that in vivo but with a lower density as compared with 18-day-old embryo and neonatal mouse. 6. In aggregates from embryonic brains 50 % of glucose is utilized by aerobic pathway. This value corresponds to glycolysis of 5-day-old rat brain, measured in incubated brain slices. 7. Although glial cells and neurones attain a relatively high degree of differentiation the structural organization of the aggregates is relatively simple. The possible factors determining the development of this structural differentiation are discussed.

Early stages of myelination in the spiral ganglion cells of the kitten during development.

Myelinated cell bodies of spiral ganglion in kittens during development can first be distinguished by light microscopic observation at the end of the first postnatal week. By mean of electron microscopic observation, the first signs of myelinated perikarya can be observed around the time of birth. Myelination is preceded by the ensheathment of the ganglion cell body by two or three layers of Schwann cell cytoplasmic processes. At birth, the first sign of myelination is visible at the basal part of the cochlea with thin thickenings which contain two or three major dense lines. At this stage, these thickenings cover only a small perikaryal area. Later on, the thickness and the number of thickenings increase and cover the major part of the cell body. The onset of myelination of the processes seems to precede the perikaryal myelination.

DEVELOPMENTAL CHANGES OF MYELIN PROTEINS IN THE CHICK PERIPHERAL NERVE

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.