Specific localization of 2',3'-cyclic nucleotide 3'-phosphohydrolase, (Ca2+/Mg2+)-ATPase, and acetylcholinesterase in human erythrocyte membrane

A procedure was developed for the detection of 2',3'-cyclic nucleotide 3'-phosphohydrolase in myelin. This assay was sufficiently to detect the low levels of 2',3'-cyclic nucleotide 3'-phosphohydrolase in human erythrocytes. The 2',3'-cyclic nucleotide 3'-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghosts and resealed ghosts were assayed for 2',3'-cyclic nucleotide 3'-phosphohydrolase (Ca2+/Mg2+)-ATPase, and acetylcholinesterase activity, and the 2',3'-cyclic nucleotide 3'-phosphohydrolase profile is the same as that of the (Ca2+/Mg2+)-ATP, an established inner membrane marker.     

Effect of the Jimpy Mutation on Expression of Myelin Proteins in Heterozygous and Hemizygous Mouse Brain

The levels of myelin basic protein, proteolipid protein, and 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) in cerebral hemispheres of wild-type, heterozygous jp/+, and hemizygous jp/Y mice of different ages were determined by radioimmunoassay and immunoblotting. In jp/Y brain the level of myelin basic protein was 8% that of wild-type at all ages. All forms of the protein were reduced although the 21.5K Mr form was relatively spared at early ages compared to the 18.5K, 17K, and 14K Mr forms. The level of 2',3'-cyclic nucleotide 3'-phosphohydrolase was 8% that of wild-type at all ages, and proteolipid protein was undetectable at any age. These results are consistent with the hypothesis that the jimpy mutation blocks myelin morphogenesis subsequent to incorporation of 21.5K Mr myelin basic protein but prior to incorporation of proteolipid protein. In jp/+ brain the levels of the three proteins were reduced commensurately to 60-70% those of wild-type. The deficit was apparent as early as 10 days after birth and remained proportionately constant throughout development. These results suggest that in jp/+ mice, X-chromosome inactivation produces a mosaic population of functionally wild-type and functionally jimpy oligodendrocytes. The former elaborate normal amounts of myelin but do not completely compensate for the myelin deficit due to the latter.     

Correlation Between 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase Activity and Demyelination In Vitro Using a Syngeneic System

Cultures of myelinated SJL/J fetal mouse spinal cord were incubated with serum and lymphoid cells from syngeneic animals with experimental allergic encephalomyelitis (EAE) induced by syngeneic spinal cord homogenate (SSCH) in complete Freund's adjuvant or others injected with complete Freund's adjuvant alone. After 24 or 48 h of exposure, demyelination was determined by light microscopic examination and quantification of 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. Cultures exposed to spleen or lymph node cells from SSCH-sensitized animals showed the greatest alterations in myelin and decreases in 2',3'-cyclic nucleotide 3'-phosphohydrolase activity whereas serum from these animals had less effect. Cells and serum from complete Freund's adjuvant-injected control animals also induced structural changes in myelin that were significantly less than changes induced by cells and serum from animals with EAE. These experiments show that lymphoid cells and serum obtained from SJL/J mice with acute EAE affected myelin biochemistry and morphology in syngeneic CNS cultures.     

Cyclic AMP Induction of the Myelin Enzyme 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in Rat Oligodendrocytes

Cyclic AMP (cAMP) is known to induce the activity of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37) in C6 rat glioma cells. This report shows that CNP is also inducible in oligodendrocytes explanted from 1-day-old rat cerebrum and grown in tissue culture. Induction was observed after a 1-day treatment with 1 mM N6, O2-dibutyryl cyclic AMP (dbcAMP) and was maximal after 5 days, reaching 200-240% of control. Induction was observed both in mixed cerebral cell cultures containing oligodendrocytes and astrocytes, and in purified cultures of oligodendrocytes prepared by a differential shakeoff procedure. Addition of dbcAMP to the cultures 3-9 days after the cells were explanted from rat brain induced CNP activity, but no induction was observed when dbcAMP treatment was begun 13 or more days after explanation. These results demonstrate that one component of myelin, CNP, is inducible in oligodendrocytes by a cAMP-mediated mechanism, and suggest a role for cAMP in the regulation of the myelin-associated functions of oligodendrocytes.     

Relation of C-6 glial cells in culture to myelin.

C-6 glial cells were shown to contain enriched in their plasma membranes an enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase, characteristic of myelin, and, in addition, proteolipid protein and two basic proteins that are identical in their electrophoretic mobilities with the respective proteins found in myelin. The data indicate that C-6 cells exhibit features of myelin-producing glia as well as astrocytes.     

The presence of 2'', 3''-cyclic AMP 3''-phosphohydrolase in glial cells in tissue culture

Abstract— T he E nzyme 2', 3'-cyclic AMP 3'-phosphohydrolase (CNP) is regarded as a marker for myelin (KURI- HARA and MANDEL, 1970) on the basis of its regional and subcellular distribution (K urihara and T sukada , 1967), its ontogenetic characteristics (KURIHARA and TSUKADA, 1968), and its behaviour in two strains of myelin-deficient mutant mice (K urihara , N ussbaum and M andel , 1969). However we have isolated highly-purified preparations of neuronal plasma membrane from rat brain synaptosomes which contain this enzyme activity (M organ , W olfe , M andel and G ombos , 1971). Two explanations of this finding are possible. The activity could be due to the presence of myelin, but this explanation is ruled out by electron microscopy and by the low level of cerebrosides in the synaptosomal plasma membrane preparations. Myelin is extremely rich in cerebrosides ( norton and A utilio , 1966). The second possibility is that the enzyme, 2', 3'-cyclic AMP 3'-phosphohyrolase, may also be found in the glial cells from which myelin is derived (B unge , B unge and P appas , 1962). To test our hypothesis that 2', 3'-cyclic AMP 3'-phosphohydrolase is not a specific marker for myelin, but is also found, in glial cells, we have examined a tumoral glial cell line maintained in myelin-free tissue culture.     

Myelin formation in dissociated cell cultures of rat embryonic cerebral hemispheres

Developing cultures of dissociated cerebral hemispheres obtained from 18-day-old embryonic rats synthesized, or activated, a myelin-related enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This increase in activity coincided with the onset of myelination. The presence of CNPase in oligodendrocytes and myelin was demonstrated using immunocytochemical staining techniques. Myelinated axons and myelin sheaths were clearly made visible by electron microscopy of 28-day-old cultures.     

A Monoclonal Antibody Raised to Corpus Callosum Extract Reacts with 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase

Monoclonal antibody against 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid-phase radioimmunoassay, immunoblot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS-PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin-specific enzyme in CNS myelin and in cells responsible for myelin production.     

Alcohol inhibits morphological and biochemical differentiation of C6 glial cells in culture

Cultures of glial cell lines (C6) were exposed to 10-micromilligram Dexamethasone which is known to cause morphological differentiation and induction of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in these cells. Ethanol in a concentration of 1.5% abolished these responses, and at 1% diminished them.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

Expression of Hyaluronectin by C-6 Glial Cells at High Density

Hyaluronectin, a brain glycoprotein that has been localized to the nodes of Ranvier in vivo and to oligodendrocytes in primary cultures of neonatal rat brain cells, was shown by using an unlabeled immunoperoxidase method to be present in C-6 glial cells grown to high density. The density-dependent expression of this glycoprotein is in accordance with the known properties of the glial stem cells, i.e., induction of differentiated properties such as 2',3'-cyclic nucleotide-3'-phosphohydrolase, glutamine synthetase, S-100 protein, and glial fibrillary acidic protein.     

Antibodies to Myelin/Oligodendrocyte-Specific Protein and Myelin/Oligodendrocyte Glycoprotein Signal Distinct Changes in the Organization of Cultured Oligodendroglial Membrane Sheets

Abstract: Cultured murine oligodendrocytes elaborate extensive membrane sheets that, unlike multilamellar myelin in vivo, allow the study of interactions between myelin proteins and cytoskeletal elements. This article describes the events that occur due to the interaction of specific antibodies with their respective antigens, myelin/oligodendrocyte-specific protein (MOSP) and myelin/oligodendrocyte glycoprotein (MOG), which are expressed uniquely by oligodendrocytes. After antibody binding, surface anti-MOSP:MOSP complexes redistribute over those cytoplasmic microtubular veins that have 2',3'-cyclic nucleotide 3'-phosphohydrolase colocalized along them. In contrast, surface anti-MOG-MOG complexes redistribute over internal myelin basic protein domains. Long-term anti-MOSP IgM exposure results in an apparent increase in number as well as thickness of microtubular structures in oligodendrocyte membrane sheets, whereas long-term anti-MOG exposure causes depolymerization of microtubular veins in membrane sheets. These data suggest that antibody binding to these two surface proteins elicits signals that have opposite effects on the cytoskeleton in oligodendroglial membrane sheets. Thus, it is possible that signals transduced via antibody binding may contribute to the pathogenesis of diseases affecting CNS myelin.     

Bulk Separation and Long-Term Culture of Oligodendrocytes from Adult Pig Brain.

Oligodendroglial proteins labeled with radioactive amino acids were subjected to one- and two-dimensional polyacrylamide electrophoresis. Bands comigrating with myelin proteins, the basic protein (MBP), the proteolipid protein (PLP), and the Wolfgram protein (WP) doublet, were detected by Coomassie Blue staining and by autoradiography. The identity of the MBP and WP in the cellular material is evidenced by immunoblotting with specific antibodies. A comparative study of myelin samples from rat and pig CNS reveals that WP can be detected immunochemically in both species. Different protein patterns, however, are observed. Three protein bands are found with antibodies against the myelin-associated glycoprotein (MAG). The high-molecular-weight component prevails in pig myelin, whereas the medium-molecular-weight component is predominant in rat myelin. Moreover, two protein bands, of molecular weights 35,000 and 33,000 (Ol 1 and Ol 2), are present in high amounts in oligodendroglial particulate material but are not detectable in myelin. These oligodendroglial characteristic proteins are not species-specific, since they are found in preparations of cat oligodendrocytes as well. Activities of cerebroside sulfotransferase (EC 2.8.2.11) are low in freshly isolated cells and increase during the first week of culture. A reverse course of enzyme activities is observed with 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37). Values reach a minimum about day 5 in culture and recover their initial values. At day 10 they remain stable until the end of the third week of the culture period.     

Ontogenetic study of a myelin-derived fraction with 2'':3''-cyclic nucleotide 3''-phosphodydrolase activity higher than that of myelin.

A membrane fraction (SN 4), prepared from developing and mature rat forebrain by hypo-osmotic treatment of microsome (microsomal-fraction)-free myelin, appears to be closely related to the myelin-like fraction. Fraction SN 4 shows 2':3'-cyclic nucleotide 3'-phosphohydrolase activity higher than that of the parent myelin. Electrophoresis reveals a multitude of bands with the Wolfgram protein as the main component.     

CNPase activity in the vertebrate retina, retinal pigmented epithelium, and choroid

The activity of the enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase, E.C.3.1.4.37) has been studied in the retina of three vertebrate species. Activity was highest in the goldfish, followed by Xenopus laevis and Rana pipiens. Also, high activity levels were found in goldfish retinal pigment epithelium and choroid, but not in the other two species. When added to in vitro culture systems, 2',3'-cyclic nucleotides were found to have no effect on goldfish cone retinomotor movement, but caused a marked inhibition of Rana pipiens rod outer segment disc membrane shedding. It is suggested that CNPase may play a role in cellular processes requiring membrane structural reorganization.     

Protein determinants of myelination in different regions of developing rat central nervous system.

Measurements of several different protein determinants correlated with the time and rate of myelination in five areas of the central nervous system are presented. The deposition of protein in the subcellular fraction corresponding to the density of adult myelin, the appearance of basic protein characteristic myelin, the change in proportions of the individual myelin proteins, the appearance and distribution of the myelin marker 2':3'-cyclic nucleotide3'-phosphohydrolase, and the results of morphological studies of purified myelin are compared. According to these various criteria, and in agreement with the morphological observations of others, myelin appears earliest in the spinal cord, then in the brain stem, and latest in the cerebral hemispheres. Multilamellar myelin was observed in the rat brain stem and spinal cord as early as 5 days of age. The relative proportion of the individual myelin proteins changed with myelin maturation in all areas, with the larger basic protein decreasing reciprocally with increase of the smaller basic protein. The proportion of Wolfgram protein also decreased with maturation. Larger proportions of the enzyme 2':3'-cyclic nucleotide 3'-phosphohydrolase were located in the microsomal fraction at early ages. During development the enzyme activity gradually became associated more with a fraction of a density corresponding to adult myelin, suggesting the presence of precursor membrane fragments in microsomal fractions in the early stages of myelination before compact myelin formation. A significant proportion of the total nucleotide phosphohydrolase activity of the homogenate could not be recovered in subcellular fraction at early ages, but the recovers of the enzyme increased with maturation and the activity was found more in the myelin fraction.     

A New Myelin-Deficient Mutant Hamster: Biochemical and Morphological Studies

Biochemical and morphological studies were done on a new trembling mutant hamster CBB. The yield of myelin from the mutant was 30 and 40% of the control at 46 and 140 days of age, respectively, but myelin composition and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activity were normal. Morphologically, about 18% of the axons were myelinated in the mutant optic nerve at 46 days of age, in which the myelinated fibers were those with larger diameters (more than 0.6 micron), while the control had a peak at 0.4 micron in diameter. The ultrastructure and thickness of compact myelin lamellae in the mutant were normal. Myelination and the structure of peripheral nerve myelin appeared normal. The results indicate that the essential defect is the delay and arrest of myelination in the CNS, which is probably caused by either a decreased rate of synthesis of myelin components in oligodendrocytes or a defect in the oligodendrocyte-axon recognition in smaller axons.     

Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain.

We used immunocytochemical staining to localize the RLM6 form of cytochrome P-450 in rat brain. Immunofluorescence staining in vibratome sections was positive in cells that resembled oligodendrocytes, which are the cells that synthesize and maintain myelin. Double immunofluorescence staining with anti-RLM6, plus mouse monoclonal antibodies (MAb) against 2',3'-cyclic nucleotide-3'-phosphohydrolase or galactocerebrosides, showed localization of each of these oligodendrocyte "markers" in the same cells as RLM6. In vibratome sections from brains of adult rats there was faint RLM6 immunostaining in some of the myelinated fibers as well as in oligodendrocytes. In paraffin sections from adult rat brains, myelinated tracts were RLM6 positive, as were oligodendrocytes and myelinated fibers in the gray matter. Oligodendrocytes were also shown to contain glucose-6-phosphate dehydrogenase. We suggest that RLM6, which is constitutive to liver, is also constitutive to brain and, via the acetone monooxygenase reaction, which also utilizes NADPH, may contribute to the conversion of ketone bodies to substrates that could provide energy for the synthesis and maintenance of myelin.     

Oligodendroglial Differentiation in Glial Primary Cultures: Requirement for Mevalonate

The oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), is a valuable marker for expression of oligodendroglial differentiation in glial primary cultures, and the inducibility of this enzyme by dibutyryl-3',5'-cyclic AMP (dBcAMP) appears to be limited to immature or developing oligodendroglia. To investigate the relationship between the induction of CNP and the sterol biosynthetic pathway, primary cultures of glia dissociated from the brains of newborn rats were maintained in 10% fetal calf serum (FCS) and exposed to 1 mM dBcAMP on day 7 in culture. Cultures so treated for either 48 h or 72 h demonstrated a three- to fourfold induction of CNP specific activity. The magnitude of this induction was not affected when the cholesterol content of the culture medium was reduced by greater than 95% by placing the cultures in 10% lipoprotein-poor serum rather than 10% FCS during the exposure to dBcAMP. Mevinolin (10 microM), a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of the sterol biosynthetic pathway, completely inhibited the induction of CNP by dBcAMP, while not affecting either the accumulation of cellular protein per flask or rate of protein synthesis. Simultaneous addition of mevalonate (20 mM) prevented the inhibition of the induction of CNP by mevinolin. However, simultaneous addition of low-density lipoprotein sufficient to increase the cholesterol content of the medium 80-fold failed to correct mevinolin's inhibition of the induction of CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glial Cell Markers in the Reeler Mutant Mouse: A Biochemical and Immunohistological Study

The glial cell contents of S100 protein, 2',3'-cyclic AMP, 3'-phosphohydrolase (CNP), isoenzyme II of carbonic anhydrase (CAII) and butyrylcholinesterase (BuChE) were biochemically determined in the cerebellum and cerebrum of the reeler mutant mouse. Astrocytes and oligodendrocytes, shown by this study, contain abnormal amounts of these components. The CAII concentration was significantly increased in the particulate fraction of the reeler cerebellum and cerebrum (by 50% and 89%, respectively). The BuChE specific activity was greatly increased in the reeler, by 120% for cerebellum and by 40% in cerebrum. In contrast, the S100 protein concentration was reduced in the reeler cerebellum by 40% and by 25% in cerebrum, while the CNP specific activity increased by 30% in the reeler cerebellum. In addition, the glial cell distribution was studied by immunohistological techniques with antibodies directed against S100 protein, glial fibrillary acidic protein (GFA) and CAII. Apparently the density of glial cells is not significantly affected. However, the Golgi epithelial cells were usually abnormally placed and their Bergmann fibres were less well developed.