A pathology-MRI study of the short-T2 component in formalin-fixed multiple sclerosis brain.

OBJECTIVE: To determine the pathologic basis of areas not exhibiting signal of the short-T2 component of the T2 relaxation distribution in MS, as studied in formalin-fixed brain. BACKGROUND: A myelin-specific MRI signal would be of great importance in assessing demyelination in patients with MS. Evidence indicates that the short-T2 (10 to 50 millisecond) component of the T2 relaxation distribution originates from water in myelin sheaths. The authors present two cases of MS in which the anatomic distribution of the short-T2 component was correlated with the pathologic findings in postmortem formalin-fixed brain. METHOD: One half of the formalin-fixed brain was suspended in a gelatin-albumin mixture cross-linked with glutaraldehyde, and scanned with a 32-echo MRI sequence. The brain was then cut along the center of the 5-mm slices scanned, photographed, dehydrated, and embedded in paraffin. Paraffin sections, stained with Luxol fast blue and immunocytochemically for 2',3'-cyclic nucleotide 3'-phosphohydrolase for myelin and by the Bielschowsky technique for axons, were compared with the distribution of the amplitude of the short-T2 component of the comparable image slices. RESULTS: The anatomic distribution of the short-T2 component signal corresponded to the myelin distribution. Chronic, silent MS plaques with myelin loss correlated with areas of absence of short-T2 signal. The numbers of axons within lesions were reduced, but many surviving axons were also seen in these areas of complete loss of myelin. CONCLUSION: In formalin-fixed MS brains the short-T2 component of the T2 relaxation distribution corresponds to the anatomic distribution of myelin. Chronic, silent demyelinated MS plaques show absence of the short-T2 component signal. These results support the hypothesis that the short-T2 component originates from water related to myelin.-1510

2',3'-cyclic nucleotide 3'-phosphodiesterase, an oligodendrocyte-Schwann cell and myelin-associated enzyme of the nervous system.

2',3'-Cyclic nucleotide 3'-phosphohydrolase (E.C. 3.1.4.37; CNPase) is a myelin-associated enzyme. In central and peripheral nervous system tissues, the enzyme is localized almost exclusively in the two cell types that elaborate myelin, the oligodendrocyte and the Schwann cell, respectively. Nonneural sources of CNPase have also been described, but they all have much lower activities than those found in brain. The freshly isolated brain enzymes appear as closely spaced doublets at approximately 46 and 48 kDa on SDS-PAGE. The primary sequence appears highly conserved between these two proteins, designated CNP1 and CNP2. Major structural differences between these two proteins are most likely due to posttranslational modifications of the enzyme itself (certainly phosphorylation, possibly others) or to alternative splicing. The primary sequences of rat and bovine brain CNPase have now been deduced from the cDNA sequences and the enzymes appear to be unique. Current research suggests that CNPase is involved in the very rapid growth of myelin membrane during early oligodendrocyte membrane biogenesis and possibly maintenance. The absolute hydrolysis specificity, yielding 2'-mononucleotides from 2',3'-cyclic substrates, strongly suggests that CNPase is a nucleic acid enzyme, possibly related to RNA metabolism.

The localization of 2':3'-cyclic nucleotide 3'-phosphodiesterase in bovine cerebrum by immunofluorescence.

The distribution of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in fixed-tissue sections and Percoll-isolated cells (oligodendrocytes) of bovine cerebrum was studied by immunofluorescent techniques. Fluorescence in tissue sections exposed to CNP antisera was restricted to oligodendrocyte-like cells. Also, Percoll-isolated cells (oligodendrocytes) were positively labeled with CNP antisera. This study has shown that CNP may be used as an antigenic marker for oligodendrocytes.

Inhibition of 2',3'-cyclic nucleotide 3'-phosphodiesterase activity from bovine, human and guinea pig brain.

The purified oligodendrocyte enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) obtained from bovine, human and guinea pig brain was inhibited by theophylline, caffeine, cupric chloride and organomercurials using a spectrophotometric assay and 1 mM 2',3'- cyclic cytidylate as the substrate. The observed inhibition by theophylline, caffeine and organomercurials was reversible.

Intracellular localization of 2',3'-cyclic nucleotide 3'-phosphohydrolase in rat oligodendrocytes and C6 glioma cells, and effect of cell maturation and enzyme induction on localization.

We investigated whether the membrane-associated myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), is localized primarily inside the cell or exposed on the cell surface of rat oligodendrocytes and rat C6 glioma cells. Determinations were made by enzyme assays of intact, viable cells vs cells broken by freezing and thawing. Assay of both oligodendrocytes and C6 cells showed that the great majority of the CNP activity was localized inside the cells. Oligodendrocytes were also tested by immunofluorescence staining of unfixed, living cells whose membranes had been made permeable to antibody by fixation. Fixed oligodendrocytes showed intense fluorescence when incubated with rabbit anti-CNP antiserum and fluorescein-conjugated second antibody whereas unfixed cells were not stained. We then tested the possible influence on CNP localization of 3 conditions known to increase CNP specific activity: maturation of oligodendrocytes in vitro during a period when CNP specific activity increases 8-fold or more; growth of C6 cultures to high cell density; and induction of CNP activity in oligodendrocytes and C6 cells by dibutyryl cyclic AMP. Under all conditions, most CNP activity was intracellular. These results show that both the catalytic and major antigenic sites of CNP are localized primarily inside the cell, and suggest an intracellular role for CNP in oligodendrocytes. The results with C6 cells also show that these cells resemble oligodendrocytes with respect to CNP localization.

Immunofluorescence demonstration of 2':3'-cyclic-nucleotide 3'-phosphodiesterase in cultured oligodendrocytes of mouse, rat, calf and human.

Previous biochemical studies have shown that high enzyme activity of 2':3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) is found in isolated myelin and oligodendrocytes. We report here the specific and intense immunofluorescence staining of cultured oligodendrocytes obtained from the brains of mouse, rat, calf and human by rabbit antiserum specific for purified bovine CNP. Astrocytes and fibroblasts present in the cultures were negative for the immunostaining. The specificity of the CNP immunoreactivity was confirmed by blocking the reaction by prior absorption of the antiserum by purified CNP.

Localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase on cultured Schwann cells.

Primary cultures of Schwann cells were labeled by indirect immunofluorescence using an antibody directed against 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Schwann cells which had been maintained in culture for 8 weeks were labeled with this antibody. Immunoblot analysis of Schwann cell homogenates revealed a single band with a molecular weight of 54,000 daltons which corresponded to a single immunoreactive polypeptide present in myelin prepared from rat sciatic nerve. The subcellular localization of CNPase was examined by fractionation of cultured Schwann cell homogenates with linear sucrose gradients. The distribution of CNPase paralleled that of 5'-nucleotidase, a putative marker for plasma membranes. These results suggest that CNPase is localized on the plasma membranes of Schwann cells and is expressed by the cells in the absence of an axonal stimulus.

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 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.

The distribution of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in the CNS of normal (+/+) and shiverer (Shi/Shi) mice.

Immunohistochemical techniques were utilized to localize the putative myelin enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in the central nervous system (CNS) of normal (+/+) and Shiverer (Shi/Shi) mice (Mus musculus). CNP appeared to be only associated with myelinated nerve fibers in the CNS (corpus callosum, subcortical white matter, caudate nucleus, cerebellum and medulla oblongata) of +/+ mice. However, little or no immunostaining was observed in the same regions of the CNS of Shi/Shi mice, although these mice have normal levels of a CNS CNP activity. Unexpectedly, oligodendrocytes (cell periphery) were not stained for CNP in either +/+ or Shi/Shi mice, although erythrocytes were immunostained.

An investigation of 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP) in peripheral blood elements and CNS myelin.

Activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) of human erythrocyte membranes was determined in the presence of various brain CNP inhibitory compounds. Also, the hydrolysis of 2':3'-cAMP and 2':3'-cCMP by CNP of human platelets and lymphocytes was confirmed by thin layer chromatography and CNP activity was measured in lymphocytes, platelets, erythrocytes and CNS myelin. Human erythrocyte CNP activity was reduced 75 percent by the organomercurial p-chloromercuriphenyl sulfonate (1 X 10(-4) M), 46 percent by thimerosal (1 X 10(-4) M) and 35 percent by cupric chloride (1 X 10(-3) M). The 2'-AMP or 2'-CMP isomer was produced, exclusively, by the hydrolysis of 2':3'-cAMP or 2':3'-cCMP, respectively, by CNP of human lymphocytes and platelets and indicates a CNP-like activity is not only present in erythrocytes and the central and peripheral nervous systems, but also platelets and lymphocytes. CNP activities of human erythrocytes, human human and rat lymphocytes and human platelets were less than 4 percent of the activity of human and bovine CNS myelin.

Isolation of a cDNA encoding a taurine transporter in the human retinal pigment epithelium.

Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify a cDNA encoding a taurine transporter in the human retinal pigment epithelium (HRPE). The coding region of a PCR product was found to be 1863 bp long, predicting a 620-amino acid protein (69,826 Da). This cDNA sequence is almost identical to those taurine transporters recently determined in the human thyroid and placenta: 12 and 1 base pair(s) different from the reported thyroid and placenta transporter clones, respectively. The injection of mRNA in vitro transcribed from the PCR product markedly increased taurine uptake in Xenopus laevis oocytes. Taurine uptake is Na+ and Cl- dependent. Unlabeled taurine, beta-alanine and gamma-amino-n-butyric acid at 100 microM inhibited the uptake of radiolabeled taurine whereas 100 microM alpha-alanine and alpha-aminoisobutyric acid did not. A kinetic study showed that taurine uptake is mediated by a single carrier system with the apparent Michaelis-Menten constant of approximately 2 microM. These results suggest that the PCR product encodes a functional taurine transporter whose characteristics are similar to those of taurine uptake observed in the original HRPE cells. A DNA encoding the reported placental transporter was made from the PCR product by site-directed mutagenesis but it was not functional in the oocyte expression. A similar RT-PCR was performed with poly (A)+ mRNA isolated from JAR human placenta choriocarcinoma cells. This PCR product was identical to that from the HRPE. In addition, the clone of the human thyroid transporter was obtained and re-sequenced. Its translation coding region was also identical to that of the PCR product from the HRPE, showing that taurine transporters are identical in the human RPE, thyroid and placenta.

Purification of rat 2',3'-cyclic nucleotide 3'-phosphodiesterase.

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37) has been isolated from rat brain myelin by chromatography on successive columns of phenyl-Sepharose CL-4B, CM-Sepharose CL-6B, and 8-(6-aminohexyl) amino-2'AMP-Sepharose 4B. From 15 g of rat brain, approximately 400 micrograms of pure CNP was obtained, with a specific activity of 1,200 (2',3'-cyclic AMP) units/mg protein. The Km of the rat enzyme was 3.7 mM, using 2',3'-cAMP as the substrate. Isoelectric focusing of the enzyme indicated a broad isoelectric range of 8.5-9.0. On SDS polyacrylamide gels, rat CNP appears as two protein bands of approximately 48,000 and 50,000 M.W., with an upper band intensity of about 1/10 that of the lower band. The relative intensities of the bands for CNP and the molecular weights correspond to the Wolfgram proteins W1 and W2 described by other investigators. The amino acid analysis of the purified rat enzyme compared favorably with reported determinations for the bovine enzyme and also with reported values for the rat Wolfgram proteins W1 and W2.

In vivo phosphorylation of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases.

2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site.

Oligodendrocyte substratum adhesion modulates expression of adenylate cyclase-linked receptors.

The molecular mechanisms of myelin formation/reformation in the central nervous system are unknown. In previous work we have demonstrated that mature oligodendrocytes (OLG) respond to a signal(s), elicited by their adhesion to a substratum, by turning on a myelinogenic metabolism. Events occurring within 24 hr of adhesion include generation of diacylglycerol, activation of protein kinase C, phosphorylation of myelin basic protein, and enhanced synthesis of myelin lipids and proteins. To elucidate the mechanism(s) of signal transduction, we have investigated whether OLG-substratum interaction influences the level of basal cAMP and the expression of receptors coupled to adenylate cyclase. By using ovine brain OLG we have found that adhesion to a polylysine-coated surface for 24 hr increased the basal level of cAMP 2-fold and altered the expression (assessed by cAMP production) of receptors coupled to adenylate cyclase. Isoproterenol (beta-adrenergic agonist) augmented cAMP from 4 to 26 pmol/mg of protein in adhering OLG but had no such effect in nonattached OLG. Adhesion of OLG was accompanied by rapid synthesis of ethanolamine plasmalogen, a class of lipids believed to be associated with beta-adrenergic receptors. Nonattached OLG responded to prostaglandin E1 with only a 3-fold stimulation in their cAMP content; in attached OLG, 6-fold stimulation was observed. In contrast, vasoactive intestinal polypeptide elicited a 3-fold increase in cAMP in nonattached OLG but, following 24 hr of attachment, OLG did not respond to vasoactive intestinal polypeptide. The increase of cellular cAMP levels was accompanied by a 2.5-fold gain in protein kinase A. OLG-substratum adhesion resulted also in phosphorylation of the OLG/myelin protein, 2',3'-cyclic nucleotide 2'-phosphodiesterase, which proved to be a substrate for cAMP and phospholipid-, Ca2+-dependent protein kinases. These findings, in conjunction with our earlier work, implicate cAMP and diacylglycerol in signaling myelinogenesis; they suggest that phosphorylation/dephosphorylation of myelin basic protein and 2',3'-cyclic nucleotide 2'-phosphodiesterase may be key processes in the cascade of events that are initiated by adhesion of OLG to a polylysine surface (possibly acting as a surrogate for axons) and culminate in the reformation of myelin.

Assignment of the human 2',3'-cyclic nucleotide 3'-phosphohydrolase gene to chromosome 17.

2',3'-Cyclic nucleotide 3'-phosphohydrolase (CNP) has been used as a general oligodendrocyte and Schwann cell marker enzyme within the nervous system and has been the intense target of a number of recent studies. In this report, we determined the chromosomal localization of the human CNP gene using PCR on two somatic cell DNA panels. PCR amplification, using four primer pairs across an intron, confirms that the CNP gene is localized to chromosome 17. We also present the complete intron sequence of the human gene usd to make the assignment. This intron contains a c----t polymorphism located at nucleotide 1215, which may be of use in mapping the CNPase gene more precisely within chromosome 17.

Differential expression of galactocerebroside, myelin basic protein, and 2',3'-cyclic nucleotide 3'-phosphohydrolase during development of oligodendrocytes in vitro.

This paper describes the differential expression and localization of myelin components within membrane sheets produced by oligodendrocytes in vitro. In double-labeling experiments using antibodies to the myelin antigens 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and galactocerebroside (GC), the two antigens were coexpressed in at least 95% of oligodendrocytes at all ages examined. A small population of relatively undifferentiated cells expressed one antigen before the other. Within the membrane sheets produced by the cultured cells, CNP and GC are distributed differently. CNP is highly concentrated in cell bodies, in a network of processes extending from the cell body into the sheets, and around the perimeter of the sheets. CNP staining cannot be detected in some areas within the body of the sheet. When present, it is of low intensity. Under our labeling conditions, GC staining is found throughout the membrane sheets, except in the network of veins which are CNP+. GC and myelin basic protein (MBP) staining are seen in similar membrane domains even though GC is a surface component while MBP resides on the cytoplasmic face. Both the timing and localization of CNP immunostaining show that CNP is as early a marker for oligodendrocytes as GC, and support the idea that CNP may play a structural role in the myelin membrane. Double-labeling studies with GC and CNP antibodies also show that the true shape of a cell and the extent of its development are not always revealed by a single antigen. The differential distribution of antigens within membrane sheets illustrates that they contain areas of structural specialization that may reflect the situation in intact myelin.

Inhibition of bovine and human brain 2':3'-cyclic nucleotide 3'-phosphodiesterase by heparin and polyribonucleotides and evidence for an associated 5'-polynucleotide kinase activity.

The present paper establishes a 5'-polynucleotide kinase activity associated with the bovine and human brain enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) in addition to known extremely high hydrolysis rates against 2':3'-cyclic nucleotides. Modulation of the enzyme activity by the addition of polyadenylate (5') and polyuridylate (5'), histone F3, myelin basic protein (MBP), and other basic molecules suggest that RNA may be the natural substrate for both enzymes. These enzymes, isolated from brain and present in very high activities in oligodendrocytes and in isolated myelin, probably have complex functions.