Microtubule alterations in cultured taiep rat oligodendrocytes lead to deficits in myelin membrane formation

The taiep rat is a myelin mutant in which hypomyelination and progressive demyelination of the CNS are accompanied by an accumulation of microtubules within oligodendrocytes. To investigate whether and how the myelin defects were caused by microtubule abnormalities, we have established a taiep oligodendrocyte culture system in which mutant cells produce abnormally high levels of tubulin and microtubule-associated proteins and exhibit myelin defects. The studies show that abnormal microtubule accumulation and tight microtubule bundles developed in the taiep oligodendrocytes, with a higher ratio of minus-end-distal to plus-end-distal microtubules in their processes. Initially, in culture, immature taiep oligodendrocytes which have higher levels of tubulin than controls extend roughly twice as much membrane sheet as controls. The membrane sheets of the mature taiep oligodendrocytes which display the microtubule accumulation, however, grew much less rapidly compared to controls. By the fifth day in culture, a majority of the taiep oligodendrocytes had ceased the expansion of their membrane sheets and in some cases the sheets retracted. The levels of the myelin proteins, proteolipid protein and myelin-associated glycoprotein, were also markedly diminished in the mature taiep oligodendrocytes. Treatment with the microtubule depolymerizing drug nocodazole prevented not only the accumulation of microtubules but also restored the normal distribution of proteolipid proteins within the taiep oligodendrocytes. These data demonstrate that myelin synthesis in the oligodendrocyte cultures relies on the formation of a normal microtubule array, and the microtubule abnormalities are directly responsible for the myelin deficit in the taiep oligodendrocytes.     

Biochemical and immunohistochemical studies with specific polyclonal antibodies directed against bovine myelin/oligodendrocyte glycoprotein

Bovine myelin/oligodendrocyte glycoprotein (MOG) was purified from a Wolfgram protein fraction of brain myelin by molecular sieving and preparative gel electrophoresis. The N-terminal sequence of this wheat germ agglutinin reacting glycoprotein was determined. Antibodies against purified MOG and synthetic N-terminal octapeptide of MOG were produced in rabbits. Respective affinity purified antibody preparations gave identical results on Western blots. Treatment with specific glycosidases indicated that the oligosaccharide chains of MOG are only of N-chain type. This glycoprotein seems to be restricted to mammalian species since it was not detected in other animal species, ranging from fish up to reptiles. Immunohistochemical investigations on rat brain sections revealed that MOG is restricted to myelin sheaths and oligodendrocytes, thus corroborating previous results obtained with the MOG 8-18C5 monoclonal antibody. Decreased staining pattern in Jimpy brain further attested its specific localization in myelin-related structures. The octapeptide site-specific antibodies were not reactive on brain sections which may be attributed to the burying of this N-terminal sequence in the membrane. These MOG polyclonal antibodies appear to be valuable tools for further studies concerning this minor glycoprotein.Abbreviations BSA bovine serum albumin - CNS central nervous system - DM-20 minor myelin proteolipid protein - MAG Myelin-associated glycoprotein - MBP myelin basic proteins - MOG Myelin/oligodendrocyte glycoprotein - OMgp Oligodendrocyte/Myelin glycoprotein - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - PeptMOG n-terminal octapeptide of MOG - PLP major myelin proteolipid protein - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecylsulphate - TBS Tris buffered saline - WPF Wolfgram protein fraction - WGA Wheat germ agglutinin     

Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.     

Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites

During vertebrate brain development, axons are enwrapped by myelin, an insulating membrane produced by oligodendrocytes. Neuron-derived signaling molecules are temporally and spatially required to coordinate oligodendrocyte differentiation. In this study, we show that neurons regulate myelin membrane trafficking in oligodendrocytes. In the absence of neurons, the major myelin membrane protein, the proteolipid protein (PLP), is internalized and stored in late endosomes/lysosomes (LEs/Ls) by a cholesterol-dependent and clathrin-independent endocytosis pathway that requires actin and the RhoA guanosine triphosphatase. Upon maturation, the rate of endocytosis is reduced, and a cAMP-dependent neuronal signal triggers the transport of PLP from LEs/Ls to the plasma membrane. These findings reveal a fundamental and novel role of LEs/Ls in oligodendrocytes: to store and release PLP in a regulated fashion. The release of myelin membrane from LEs/Ls by neuronal signals may represent a mechanism to control myelin membrane growth.     

Wheat germ agglutinin fracture-label of Golgi apparatus membranes in proliferating cells

The thin section fracture-label technique has been recently used to analyze the distribution and compartmentalization of fully glycosylated components on intracellular membranes. Labelling with the lectin wheat germ agglutinin over the freeze-fractured membranes of Golgi apparatus in various secretory and non-secretory cells as well as in human peripheral lymphocytes was always very weak or absent even over the trans-most cisternae. In order to investigate if the labelling density may reflect the cellular activity in membrane biogenesis, we used in this study wheat germ agglutinin fracture-label of rapidly proliferating cells and mitogen-activated lymphocytes. Labelling over the fractured cisternae of the medial and trans portions of the Golgi apparatus was intense. Treatment with cycloheximide of proliferating cells induced a drastic reduction of the labelling over the Golgi cisternae.     

In vitro insertion of the myelin proteolipid apoprotein into oligodendrocyte plasma membranes

Uncoated vesicles (UCV) loaded with the myelin proteolipid apoprotein covalently tagged with fluorescein (PLP_F) were found to interact with isolated oligodendrocytes from bovine brain at 4°C as well as at 37°C. After 1.5 hours of incubation, the labeled protein was localized in the cell membranes. After 2.5 hours the fluorescence intensity associated with the oligodendrocytes decreased and completely disappeared at t=3.5 hours. Addition of KCl or EDTA in the incubation medium significantly hindered the interaction with cells. In contrast, the elimination of membrane proteins from UCV did not perturb cell labeling. A specific role of PLP was suggested since UCV loaded with a soluble protein (BSA_F) led to a weak cell labeling.Abbreviations IAF 5-iodacetamidofluorescein - BSA bovine serum albumin - BSA BSA labelled with IAF - PLP proteolipid apoprotein - PLP_F aqueous form of PLP tagged with IAF - CV coated vesicles - UCV uncoated vesicles - UCV^*PLP_F UCV loaded with PLP_F - MV model vesicles This work was suported by Cnrs and INSERM.     

Microtubule disruption does not prevent intracellular transport and secretory processes of cultured fibroblasts

The effect of microtubule disruption on intracellular protein translocation and secretion of cultured human fibroblasts was studied. Experiments with fluorochrome-labeled wheat germ agglutinin showed that treatments with demecolcine or vinblastine sulfate rapidly brought about dispersal of Golgi organization. Similarly, monoclonal tubulin antibodies revealed in immunofluorescence a complete disappearance of microtubules under these conditions. Cells exposed to demecolcine or vinblastine sulfate appeared to secrete both fibronectin and other polypeptides similar to the control cells, as judged by electrophoretic analysis of the culture medium. In line with this, immunofluorescence studies of the cells, treated with the antimitotic drugs and then exposed to puromycin, showed a rapid depletion of intracellular fibronectin and collagen type III-specific staining. On the contrary, cells exposed first to monensin and then to demecolcine or vinblastine sulfate and puromycin, showed an accumulation of these proteins in vesicles in the Golgi region. The results suggest that in cultured fibroblasts microtubules do not have a direct permissive role in the intracellular translocation of the secreted proteins although they are involved in the maintenance of the Golgi apparatus. The secretory processes, however, appear to continue also in cells with dispersed Golgi organization and lacking microtubules.     

Developmental expression of MOSP in cultured oligodendrocytes

Myelin/oligodendrocyte specific protein (MOSP) is a recently characterized 48 kDa surface membrane protein that is expressed exclusively by oligodendrocytes in the CNS. In this report, evidence is presented for the identification of the stage in the oligodendrocyte lineage when MOSP is first expressed. MOSP initially appears on immature oligodendrocytes about four to five days postnatal, which is about one to two days after the appearance of galactocerebroside and sulfatide. The initial expression of MOSP occurs at the stage in development when oligodendrocytes are elaborating processes and just beginning to form membrane sheets. Since 1) MOSP is capable of signaling increases in microtubular structures in oligodendrocytes and 2) microtubular structures may be essential for extension of growing processes and the formation of membrane sheaths, MOSP may play an important role in differentiation of oligodendrocytes and the formation of myelin.Special issue dedicated to Dr. Marjorie B. Lees.     

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.     

Purification and immunohistochemical localization of rat brain myelin proteolipid protein.

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N-terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X-100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10-day-old rats.     

The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP''s diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.     

Isolation and properties of gamma protein,the major transmembrane sialoglycoprotein of the HeLa cell

The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [³H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [¹²⁵I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [¹²⁵I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

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.     

Effects of wheat germ agglutinin on membrane transport

(1) Low concentrations of wheat germ agglutinin are cytotoxic toward several tissue culture lines, including Chinese hamster ovary cells, Swiss 3T3 cells, mouse L cells and baby hamster kidney cells. The LD50 ranged from 1 to 5 microgram wheat germ agglutinin per ml. Similar concentrations of the lectin inhibited the transport of the non-utilizable amino acids alpha-aminoisobutyric acid and cycloleucine and inhibited the uptake of thymidine. In contrast, 2-deoxy-D-glucose uptake was not altered and colchicine uptake was enhanced. (2) The inhibition of alpha-aminoisobutyric acid uptake occurred within minutes after lectin addition and was maximal by 1 h. Maximal inhibition ranged from 50 to 70% of control values. Studies of the kinetics of the uptake demonstrated that wheat germ agglutinin decreased the V of the uptake by 70% without affecting the apparent Km. Ovomucoid, a haptene inhibitor of wheat germ agglutinin-binding to cell surface receptors, prevented the wheat germ agglutinin-induced inhibition of alpha-aminoisobutyric acid transport. Three other lectins (Concanavalin A, Phaseolus vulgaris E-phytohemagglutinin and L-phytohemagglutinin) inhibited the uptake by 20% or less at doses up to 50 microgram/ml. (3) We propose that the cytotoxicity of wheat germ agglutinin probably results in part, if not totally, from membrane alterations which impair multiple membrane transport systems.     

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.