Myelin-associated glycoprotein (MAG) in the CNS of adult shiverer (Shi/Shi) mice

Brain fractions of adult control (+/+) and shiverer (Shi/Shi) mice were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Immunostaining with specific antisera against rat brain myelin-associated glycoprotein (MAG) was detected at about the 96-kD region of gels in electrophoresed samples of the total homogenate, low-speed supernatant fraction, and low- and high-speed sedimentable portions of brain from +/+ mice. Reduced immunostaining was observed in the corresponding samples of brain fractions from Shi/Shi mice. The cerebrum, cerebellum, and medulla of +/+ and Shi/Shi mice were examined immunocytochemically for MAG on paraffin-embedded sections. Periaxonal immunostaining for MAG was observed in all the regions and the highest concentrations were in the corpus callosum, in the central cores of cerebellar folia, and in the medulla. Patterns of distribution were similar in +/+ and Shi/Shi mice, although the density of immunostaining around individual axons and the number of immunostained axons were significantly reduced in Shi/Shi mice. In addition, the three brain regions of Shi/Shi mice exhibited oligodendrocyte-like cells that contained immunostaining for MAG in the cytoplasm and periphery of their perikarya. This type of immunostained cell was not observed in +/+ mice. In this study, immunoblotting with brain fractions and immunocytochemistry revealed strong evidence for reduced concentrations of MAG in the CNS of Shi/Shi mice compared to control mice. In addition, there is immunocytochemical evidence for abnormal accumulation of MAG in perikarya of oligodendroglial-like cells, suggesting the possibility of a transport block for myelin proteins in the shiverer mutant.     

Distribution of myelin-associated enzymes and myelin proteins into membrane fractions from the brains of adult shiverer and control (+/+) mice

The Shiverer, an autosomal recessive mutation in the mouse, is characterized by a severe deficiency in CNS myelin. The concentrations of the myelin basic and proteolipid proteins in the brain of two-month-old shiverer mice, although high enough to be measured, were much lower than in the control (+/+) brains. In contrast, the specific acitivies of teh myelin-associated enzymes, 2′, 3′-cyclic nucleotide-3′-phosphohydrolase (CNP), 5′-nucleotidase, and carbonic anhydrase, were close to normal in the brains of the mutants. The activities of these enzymes and the concentrations of the myelin large basic and proteolipid proteins were compared in membrane fractions prepared, by differential and density gradient centrifugation, from the brains of shiverer and +/+ control mice. In myelin purified from the brains of shiverer mice the specific activities of 5′-nucleotidase and CNP were close to normal, and the specific activities of all three enzymes were normal in a crude myelin fractions from brains of the mutants. However, in the shi/shi brains abnormally high proportions of the three myelin-associated enzymes were distributed into the P₃ (microsomal) fraction and into membrane fractions denser than myelin. The major myelin proteins, although low in total amounts in the mutants' brains, were distributed into the membrane fractions from control and shiverer brains in relative proportions similar to the relative proportions observed for the three enzymes. Thus, carbonic anhydrase, 5′-nucleotidase and CNP in the brains of shiverer mice are not truly dissociated from the major myelin proteins but are, rather, distributed for the most part into the same populations of membranes as are the residual, small amounts of the myelin basic and proteolipid proteins.     

Comparison of the distribution of Na, K-ATPase and myelin-associated glycoprotein (MAG) in the optic nerve, spinal cord and trigeminal ganglion of shiverer (shi/shi) and control (+/+) mice

Na+,K+ ATPase and myelin-associated glycoprotein (MAG) were studied by immunocytochemistry on paraffin sections of the spinal cord, optic nerve and trigeminal ganglion of adult control (+/+) and CNS myelin-deficient shiverer (shi/shi) mice. Immunostaining for Na+, K+-ATPase outlined the periphery of nerve fibers in the spinal cord white matter, optic nerve and trigeminal ganglion of +/+ and shi/shi mice. Immunostaining for Na+,K+-ATPase appeared somewhat denser in the optic nerve and spinal cord lateral funiculi of shi/shi than in +/+ mice. In addition, immunostaining for Na+,K+-ATPase was demonstrated at the plasmalemma of presumed satellite cells situated at the periphery of ganglion cell bodies in the trigeminal ganglion of both species of mice. Immunostaining for MAG was localized along the periphery of nerve fibers in the spinal cord funiculi (with little immunostaining within gray horns), optic nerve and trigeminal ganglion of both +/+ and shi/shi mice. The major differences between shi/shi and +/+ mice were that the number of MAG-immunostained nerve fibers was greatly reduced in the spinal cord funiculi and the density of immunostaining was slightly increased in the optic nerve of shi/shi mice. The numbers of MAG-immunostained nerve fibers in trigeminal ganglion were similar in both species. Also, the cytoplasm of some oligodendrocyte-like cells was found densely immunostained for MAG in the spinal cord and optic nerve of shi/shi mice, but not of +/+ mice. This light microscopic study provides evidence that the defective shiverer gene leads to a decrease in MAG deposition and to aggregations of MAG-like material within perikarya of oligodendrocyte-like cells in regions of the CNS.     

3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution

Knowledge of the distribution and function of the vanilloid receptor (VR-1 or TRPV1) in the CNS lacks the detailed appreciation of its role in the peripheral nervous system. The radiolabelled vanilloid agonist [3H]resiniferatoxin (RTX) has been used to indicate the presence of TRPV1 receptor protein in the brain but low specific binding has complicated interpretation of this data. Recently, support for a more widespread CNS distribution of TRPV1 mRNA and protein has been provided by RT-PCR and antibody data. We have exploited the availability of TRPV1 null mice and used [3H]RTX autoradiography in the CNS of TRPV1 wild-type and TRPV1 null mice to identify the component of [3H]RTX binding to TRPV1 receptor protein. In the brains of TRPV1+/+ mice, specific [3H]RTX binding was broadly localised with the greatest binding in the olfactory nuclei, the cerebral cortex, dentate gyrus, thalamus, hypothalamus, periaqueductal grey, superior colliculus, locus coeruleus and cerebellar cortex. Specific binding was also seen in the spinal cord and sensory (dorsal root and trigeminal) ganglia. This binding was much lower but not abolished in most regions in the TRPV1-/- mice. Nonspecific binding was low in all cases. The present study unequivocally demonstrates a widespread and discrete distribution pattern of the TRPV1 receptor protein in the rat central nervous system. The presence of TRPV1 receptors in several brain regions suggests that it may function as a cannabinoid-gated channel in the CNS.     

Monoclonal antibody production to human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2

Monoclonal antibodies against human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.     

Four-kilobase sequence of the mouse CNP gene directs spatial and temporal expression of lacZ in transgenic mice

The gene encoding 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5′-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, β-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus—tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5′-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination. J. Neurosci. Res. 53:393–404, 1998. © 1998 Wiley-Liss, Inc.     

Shiverer*jimpy double mutant mice. I. Biochemical evidence for reciprocal intergenic suppression

Shiverer and jimpy are neurological mutations that cause hypomyelination in the mouse CNS. The 3 major protein components of CNS myelin are: myelin basic protein (MBP), proteolipid (PLP) and 2', 3'-cyclic nucleotide phosphohydrolase (CNP). Previous work has shown that in jimpy animals the CNS contains reduced levels of MBP and CNP while PLP is undetectable. In shiverer animals the major forms of MBP ae undetectable in either the CNS or PNS, but the level of CNP is unaffected by mutation. In this study we have measured MBP, PLP and CNP in both the CNS (cerebral hemispheres) and PNS (sciatic nerve) of mice carrying the jimpy and shiverer mutations, individually and in combination. The results indicate that in the double mutant the levels of all 3 myelin proteins in both the CNS and PNS are intermediate between the levels in jimpy and the levels in shiverer animals. This means that part of the biochemical phenotype of the jimpy mutation (reduced levels of CNP and absence of PLP) is suppressed by the shiverer mutation, and part of the biochemical phenotype of the shiverer mutation (absence of MBP) is suppressed by the jimpy mutation. Possible mechanisms for this reciprocal intergenic suppression are discussed.     

Isoprenoid modification permits 2',3'-cyclic nucleotide 3'-phosphodiesterase to bind to membranes.

The myelination-related enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a relatively abundant protein in the CNS possesses the C-terminal isoprenylation consensus domain found in a small family that includes the ras oncoproteins and their relatives, some G-proteins, and nuclear lamins. We found that CNP, like these other proteins, is modified posttranslationally by an isoprenoid derived from mevalonic acid. It appears that only the smaller of the two CNP isoforms (CNP1) is isoprenylated, but similar modification of CNP2 cannot be excluded. Inhibition of isoprenoid synthesis by Lovastatin blocks the binding of newly synthesized CNP to cell membranes; binding is restored upon addition of mevalonate to the culture medium. This shows that isoprenylation is permissive for the well-known avid association of CNP with membranes.     

Membrane-bound 2'', 3'',-cyclic nucleotide 3''-phosphohydrolase activity of lymphocytes, granulocytes and erythrocytes in multiple sclerosis

A sensitive fluorimetric method was used for the assay of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity of lymphocyte, granulocyte and erythrocyte membranes from patients with multiple sclerosis (MS). The data obtained were compared with the corresponding data from normal individuals. CNP activities of granulocyte and erythrocyte membranes of the 2 groups did not differ significantly. However, a 40% decreased membrane CNP activity of MS lymphocytes was found when the data were compared with the normal lymphocytes' activity (P less than 0.0005) by both the non-parametric median test and Student's t-test. A role of CNP in immunoregulation is suggested.     

Early ultrastructural defects of axons and axon–glia junctions in mice lacking expression of Cnp1

Most axons in the central nervous system (CNS) are surrounded by a multilayered myelin sheath that promotes fast, saltatory conduction of electrical impulses. By insulating the axon, myelin also shields the axoplasm from the extracellular milieu. In the CNS, oligodendrocytes provide support for the long‐term maintenance of myelinated axons, independent of the myelin sheath. Here, we use electron microscopy and morphometric analyses to examine the evolution of axonal and oligodendroglial changes in mice deficient in 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) and in mice deficient in both CNP and proteolipid protein (PLP/DM20). We show that CNP is necessary for the formation of a normal inner tongue process of oligodendrocytes that myelinate small diameter axons. We also show that axonal degeneration in Cnp1 null mice is present very early in postnatal life. Importantly, compact myelin formed by transplanted Cnp1 null oligodendrocytes induces the same degenerative changes in shiverer axons that normally are dysmyelinated but structurally intact. Mice deficient in both CNP and PLP develop a more severe axonal phenotype than either single mutant, indicating that the two oligodendroglial proteins serve distinct functions in supporting the myelinated axon. These observations support a model in which the trophic functions of oligodendrocytes serve to offset the physical shielding of axons by myelin membranes. © 2009 Wiley‐Liss, Inc.     

Molecular cloning and characterization of rat brain 2′, 3′ -cyclic nucleotide 3′ -phosphodiesterase isoform 2

We have isolated a cDNA coding for the larger isoform of the rat brain 2′,3′ -cyclic nucleotide 3′ -phosphodiesterase (CNP2), a protein associated with myelination in the central nervous system (CNS). The complete 420 amino acid sequence was deduced from the nucleotide sequence of the cDNA. Sequence comparisons show that rat CNP shares 96% homology with mouse, 84% with bovine, and 86% with human CNP. Errors in the published sequence of rat CNP1 have now been corrected. Comparisons with other proteins reveal several interesting conserved motifs, including two leucine repeat heptads, and two consensus motifs for phosphorylation in the N-terminal domain of CNP2.© 1994 Wiley-Liss, Inc.     

Comparative immunocytochemistry of Pelizaeus-Merzbacher disease, the jimpy mouse, and the myelin-deficient rat

Patients with Pelizaeus-Merzbacher disease (PM), hemizygous mice with the jimpy mutation (jp/Y), and hemizygous rats with X-linked myelin deficiency (md/Y) share a profound lack of proteolipid protein (PLP) in their central nervous systems (CNS). The peripheral nervous system is normal. These X-linked disorders are associated with or actually caused by the lack of normal oligodendrocytes. Vibratome sections of brain were incubated with antisera to myelin basic protein (MBP), myelin-associated glycoprotein (MAG), 2':3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) (EC 3.1.4.37), PLP, a synthetic PLP-peptide, glial fibrillary acidic protein (GFAP), and transferrin. Reaction product was developed by sequential incubation with biotinylated second antibodies, the avidin-biotin-peroxidase complex (ABC), and diaminobenzidine (DAB) plus hydrogen peroxide as chromogenic substrates. In PM, jp/Y and md/Y, islands of myelin-like structures were revealed by antisera to MBP, MAG, and CNP. Reaction product after application of anti-PLP was absent. Reaction product after anti-PLP-peptide was restricted to infrequent bizarre cells possibly representing abnormal oligodendroglia. The lack of oligodendrocytes in jp/Y and md/Y could also be confirmed by immunocytochemistry for transferrin.     

Central myelin in the first hybrid mice produced by intercrossing homozygotes of shiverer and myelin-deficient mutants

The first hybrid mice ('shiverer*mld' mice) produced by intercrossing the homozygotes of the shiverer (BALB/c strain) and mld (MDB/Dt strain) were used for investigating the fine structure of the myelin lamellae, immunoreactive pattern for myelin basic proteins (MBP) and Golgi impregnated images of oligodendrocytes, with special reference to the influence of aging. All of the hybrid mice had an intermediate coat color between the white of the shiverer and black-brown of the mld, and revealed the same neurological symptoms, intention tremor, ataxic behavior, etc., as those of the shiverer and mld. The central myelin lamellae of the 'shiverer*mld' mouse exhibited the similar characteristics to the shiverer type rather than the mld type from the standpoint of the infrequent occurrence of major dense lines, although they did display a tendency to increase major dense lines with aging like the mld. Observation of the immunohistochemical preparations for MBP showed that immunopositive myelin sheaths were present in the white matter, although they were far more infrequent than those of the mld mutant, probably reflecting the amount of major dense lines. Thus, in the CNS of the 'shiverer*mld' mouse, the MBP-synthesis was possibly much more disturbed than in the mld mutant, or at least, revealed an intermediate pattern between the mld and shiverer.     

An immunochemical investigation of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in bovine cerebrum and human oligodendroglioma

Bovine cerebrum, including the corpus callosum, and a human oligodendroglioma were investigated by an immunohistochemical technique to determine the distribution of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Also, three human oligodendrogliomas (ODG) were characterized by an immunoblot procedure to identify a protein(s) with cross-reacting determinants to CNP and assayed for CNP activity. CNP was localized to oligodendrocytes in the corpus callosum and subcortical white matter and gray matter. Also, nerve fibers appeared to be stained. Further, cells of the human oligodendroglioma were immunostained which were similar in morphology to those cells stained in the bovine cerebrum; however, fewer than 5% of the oligodendroglioma cells were immunostained. Immunoblotting revealed two separate and distinct bands for the three oligodendrogliomas, showing cross-reactivity to bovine CNP antisera at about 53,000 and 46,000 daltons. Specific CNP activity of the three human oligodendrogliomas ranged from 0.4 to 1.6 mumole of 2':3'-cAMP hydrolyzed/min/mg protein.     

Early adrenalectomy stimulates subsequent growth and development of the rat brain

Rats were adrenalectomized (ADX) or sham-operated (SHAM) on the 11th day of life and subsequent brain development (cerebrum and cerebellum) studied in terms of tissue weight and biochemical composition. Measured at about 65 days of age, early ADX subjects had significantly heavier brains (in terms of both wet and dry weights) than SHAMs, despite being lighter in overall body weight. Brain protein and DNA contents were elevated, as was the activity of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP), a myelin marker enzyme. Glycerol-3-phosphate dehydrogenase (GPDH), a glucocorticoid-regulated enzyme, was reduced in activity. Although both the cerebrum and cerebellum showed the growth-enhancing effects of early adrenalectomy, the DNA and CNP changes were most pronounced in the cerebrum. Finally, the effect of adrenal removal on myelinogenesis was confirmed by subcellular fractionation experiments demonstrating that more myelin could be recovered from the brains of ADX than from SHAM animals. These results are significant in terms of the influence of adrenal secretions on normal brain development and the role of GPDH in myelin lipid biosynthesis.     

Developmental expression of 2',3'-cyclic nucleotide 3'-phosphohydrolase in dissociated fetal rat brain cultures and rat brain

The development in primary dissociated rat brain cultures of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity, the accumulation of CNP protein, and the number of cells accumulating this protein have been quantitatively determined as a function of time in culture. Parallel determinations have been made for the first two parameters for developing rat brain. The developmental profile of CNP enzymatic activity and amount of CNP protein in culture paralleled that observed in rat brain, in which the period of most active development occurred 7-25 days after birth. Mean CNP activities of 5.6 and 8.1 mumol/min/mg total protein were recorded for the cultures and rat brain, respectively, at their maximal levels. The corresponding mean values for the CNP protein accumulation were calculated to be 138 and 150 pmol/mg total protein, respectively. Thus maximal specific activities of the CNP protein were estimated to be about 800 and 1,000 mumol/min/mg CNP protein for culture and rat brain enzyme, respectively. Approximately three million cells expressing CNP appeared in the cultures per dissociated fetal rat brain seeded. Each CNP+ oligodendrocyte in culture had an average CNP activity of 3.2 pmol/min, and an average CNP protein content of 0.09 fmol (5.4 X 10(7) molecules), values which remained nearly constant during the course of development. Two principal conclusions are drawn from these data. First, the dissociated fetal brain culture system reproduces rather accurately the temporal developmental pattern of CNP expression occurring in the rat brain, but some important quantitative differences occur which suggest the need for additional environmental stimuli missing in these cultures. Second, the quantitative increases in CNP specific activity and amount of CNP protein occurring during oligodendrocyte differentiation in these cultures are primarily the result of increases in the number of CNP+ cells present which upon differentiation express very quickly, via an off-on regulation, steady-state levels of the enzyme.     

Characterization of a novel monoclonal anti-myelin basic protein antibody: use in immunoblotting and immunohistochemical studies

Monoclonal antibodies (MAbs) to the myelin basic protein (MBP) were produced in CAF1 (BALB/c x A/J) mice immunized with intact bovine MBP. A number of MAbs were obtained, one of which was characterized in detail with respect to its isotype, antigenic determinant on the MBP, the spectrum of antigens with which it reacted in mouse brain, and its immunohistochemical staining characteristics. This monoclonal, GB-1 (an IgG1), recognized an epitope within residues 30-51 of bovine MBP. It also reacted with a family of MBP-related proteins present in brain homogenates of mice from 7-35 days. Immunohistochemically, GB-1 stained myelinated fibers and oligodendrocytes in the rodent CNS. A second monoclonal (GB-2, and IgM) was partially characterized. It reacted with intact MBP when it was immobilized to plastic or nitrocellulose, but it was not found to be useful for immunoblots or immunohistochemistry.     

Poor myelination in the central nervous system of "dilute-lethal mutant mice" (d1/d1)

The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), as a marker for myelinogenesis, was measured in different parts of the nervous system of dilute-lethal mutant mice (d1/d1). The activity in terms of micromoles of 2'-AMP formed per milligram of protein of homogenate per minute was found to be exceedingly reduced in the cerebrum, brain stem, and medulla oblongata, slightly reduced in the cerebellum and cervical spinal cord, but not reduced in the thoracic spinal cord and the optic and sciatic nerves. These results clearly indicate that dilute-lethal mutant mice show poor myelination in the cerebrum, brain stem, and medulla oblongata.     

Oligodendrocytes but not astrocytes express apolipoprotein E after injury of rat optic nerve

Apolipoprotein E (apoE), a lipid-binding glycoprotein involved in transport and metabolism of phospholipids and cholesterol, is synthesized and secreted at elevated rates following transection of mature rat peripheral and central nerves. In the peripheral nervous system (PNS) infiltrating macrophages express apoE during Wallerian degeneration. Following injury of the optic nerve (ON) apoE synthesis is significantly stimulated but the apoE-expressing cells have thus far not been identified. This study used 1 micron and thin cryosections to identify the cellular source of apoE in transected ON. Serial 1 micron frozen sections were stained by avidin-biotin-peroxidase complex immunocytochemistry by using a specific antiserum to apoE and by antibodies that identify different cell types: glial fibrillary acidic protein (GFAP) for astrocytes, 2',3'-cyclic nucleotide-3' phosphodiesterase (CNP) for oligodendrocytes, and ED1 for macrophages. In normal ON both astrocytes and oligodendrocytes were apoE-positive. One week after ON transection oligodendrocytes accounted for the majority of apoE-positive cells, while apoE immunoreactivity had disappeared from astroglial cell bodies and processes. In contrast to the PNS only a few ED1/apoE-positive macrophages were present in ON 7 days after transection. By using immunogold-labeled ultrathin cryosections apoE could be localized in the Golgi apparatus of oligodendrocytes, indicating synthesis by these cells. Our data suggest that oligodendrocyte-derived apoE protein may participate in the redistribution of myelin lipids after CNS injury.