Effects of experimental hypothyroidism on myelin sheath structural organization

A previous study using the 2′3′cyclic nucleotide 3′phosphodiesterase (CNPase), an oligodendroglial marker that also stain ensheathed fibers, showed a decrease in the number of immunoreactive fibers and a change in the pattern of CNPase immunoreactivity (CNPase+) in hypothyroid animals. CNPase+ fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls. As CNPase, in adult animals, can be found only in regions in which oligodendrocyte cytoplasm remains as internal, external and paranodal loops, it was suggested that the reduction of thyroid hormone levels leads to a failure in myelin compaction. Previous data showed a higher frequency of some abnormalities in myelin sheath as multiple cytoplasmic loops and redundant myelin profiles in mutant animals that present a failure in myelin compaction. The increase in the frequency of these abnormalities (multiple internal and external loops and redundant myelin) indicates a failure in the interrelations between the axons and the oligodendroglial processes. To verify if the thyroid hormone deficiency during CNS development disturbs these interrelations, we evaluated the frequency of the morphological abnormalities (multiple internal and external loops and redundant myelin) in myelin sheath of corpus callosum (cc) in experimental hypothyroidism. Randomic fields were kept by electron microscopy and the analysis of the frequency of morphological abnormalities showed a significant difference in hypothyroid animals at 60-day-old (PND60), with no significant differences at 90-day-old (PND90) animals. The frequency of multiple internal loops is higher in hypothyroid animals at PND60 that indicates a disturbance in the wrapping by the oligodendroglial process. These findings showed that thyroid hormone might modulate the axon-oligodendroglial relationships that are important for the adequate temporal sequence of events that occur during myelinogenesis, with possible consequences on myelin compaction.     

Jimpy Mice: Quantitation of Myelin-Associated Glycoprotein and Other Proteins

Myelin-associated glycoprotein (MAG), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity, myelin basic protein (BP), and proteolipid protein (PLP) were quantitated in the brains of 20-day-old Jimpy and control mice. The levels of MAG, CNPase, and BP in Jimpy brains were 5.3%, 9.7%, and 1.9% of those in control brains, respectively. Immunoblotting analysis did not reveal an increased apparent Mr for MAG in the Jimpy mouse, as has been observed in some other hypomyelinating murine mutants. PLP was reduced more than the other proteins, as it was not detected by an immunoblotting technique that was capable of detecting 0.5% of the control level.     

Myelin-Associated Glycoprotein in the Central and Peripheral Nervous System of Quaking Mice

The myelin-associated glycoprotein (MAG) was quantitated in the CNS and PNS of quaking mice and the levels compared to the levels of myelin basic protein (MBP) and 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. In the brainstems of 36-day-old quaking mice, MBP, MAG, and CNPase were reduced to 12, 16, and 29% of control levels, respectively. In the sciatic nerves of the 36-day-old quaking mice, MBP and CNPase were 38 and 75% of control levels, respectively, whereas the concentration of MAG was unchanged or slightly increased. Similar quantitative results were obtained for the sciatic nerves and spinal roots of 7-month-old quaking mice. Immunoblots showed that the principal MAG band from the brainstems, sciatic nerves, and spinal roots of the quaking mice had a higher than normal apparent Mr. In addition, there was a minor component reacting with anti-MAG antiserum in the brainstems of the quaking mice that had a slightly lower Mr than control MAG and was not detected in the normal mice. The results for the quaking mice are compared with those from similar studies on other mutants with dysmyelination of the CNS and PNS.     

Alteration of 5'-Nucleotidase and Na+, K+-ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (jimpy, quaking, Trembler, shiverer, and mld). Comparison with CNPase in the Developing Sciatic Nerve from Trembler

Abstract: 5'Nucleotidase and Na⁺,K⁺-ATPase are very probably myelin-associated enzymes, although not specific for this membrane. Thus, it is important to determine their activity in dysmyelinating mutants in either CNS (quaking, jimpy, shiverer, and mld) or PNS (Trembler). CNS: The activity of 5'nucleotidase was lower in mouse than in rat (10.5 and 28.0 nmol/min/mg protein in brain, respectively). In mouse myelin, the activity was 30 nmol/min/mg protein (and 72 in rat myelin). In mutants, the brain activity was very close to normal. In contrast, ATPase, the activity of which was higher in myelin as compared with forebrain homogenate, presented a reduced activity in various 21-day-old and adult mutants, except Trembler. It was normal in 8-day-old quaking and in cerebella from mutants. PNS: ATPase was lower than in brain and reduced in most mutants, this being expected for Trembler and quaking but not for shiverer and mld. 5'-Nucleotidase activity was higher compared with that in brain homogenate (relatively stable between 10-day postnatal and adult). It was affected in the mutants; in Trembler it was nearly normal in young animals but increased during development. Thus in Trembler, two different myelin-related enzymes and a myelin-specific enzyme (CNPase) presented different developmental patterns: ATPase was always reduced, 5'-nucleotidase was normal, and CNPase was slightly below normal in young (68% of the control value); CNPase activity declined during development but 5'-nucleotidase increased (42% and 190% of the control in 60-day-old animals). It is necessary to consider these results in parallel with alterations in the PNS because of Schwann cell abnormalities. Thus, determination of these two enzymes will provide a useful tool to study myelination and myelin assembly under both normal and pathological conditions.     

Differential expression of 2′∶3′-cyclic nucleotide 3′-phosphodiesterase in cultured central,peripheral, and extraneural cells

The relative levels of the central nervous system myelin marker enzyme 2:3-cyclic nucleotide 3-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C₆ and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme.     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

Age-dependent myelin degeneration and proteolysis of oligodendrocyte proteins is associated with the activation of calpain-1 in the rhesus monkey

Myelin provides important insulating properties to axons allowing for propagation of action potentials over large distances at high velocity. Disruption of the myelin sheath could therefore contribute to cognitive impairment, such as that observed during the normal aging process. In the present study, age-related changes in myelin, myelin proteins and oligodendrocyte proteins were assessed in relationship to calpain-1 expression and cognition in the rhesus monkey. Isolation of myelin fractions from brain white matter revealed that as the content of the intact myelin fraction decreased with age, there was a corresponding increase in the floating or degraded myelin fraction, suggesting an increased breakdown of intact myelin with age. Of the myelin proteins examined, only the myelin-associated glycoprotein decreased with age. Levels of the oligodendrocyte-specific proteins 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin/oligodendrocyte-specific protein (MOSP) increased dramatically in white matter homogenates and myelin with age. Age-related increases in degraded CNPase also were demonstrable in white matter in association with increases in activated calpain-1. Degraded CNPase was also detectable in myelin fractions, with only the floating fraction containing activated calpain-1. The increases in the activated enzyme in white matter were much greater than those found in myelin fractions suggesting a source other than the myelin membrane for the marked overexpression of activated calpain-1 with age. In addition, CNPase was demonstrated to be a substrate for calpain in vitro. In summary, changes in myelin and oligodendrocyte proteins occur with age, and they appear to have a significant relationship to cognitive impairment. The overexpression of CNPase and MOSP suggests new formation of myelin by oligodendrocytes, which may occur in response to myelin degradation and injury caused by proteolytic enzymes such as calpain.     

Catabolic Regulation of the Expression of the Major Myelin Glycoprotein by Schwann Cells in Culture

Previous studies have suggested that neonatal Schwann cell cultures deprived of axonal contact do not express components of the myelin membrane, including the major myelin glycoprotein, P0. In contrast, Schwann cells from permanently transected, adult nerve exhibit continued biosynthesis of P0 after culture, suggesting that the ability to express the myelin glycoprotein may depend on the degree of cellular differentiation. To examine further the ability of Schwann cell cultures to express P0 as a function of age, we have performed precursor incorporation studies on endoneurial explants from 4- to 12-day-old rat sciatic nerves after 5 days in culture. The data reveal that explants from 12-day-old animals synthesize detectable levels of this integral myelin protein when assayed by [3H]mannose incorporation, even though there is no apparent myelin assembly in the cultures. Pulse-chase analysis of cultures from 12-day-old rats demonstrates that [3H]mannose-labeled P0 is substantially degraded within 3 h. This catabolism largely can be prevented by the addition of swainsonine, ammonium chloride, or L-methionine methyl ester to the pulse-chase media. The former agent alters oligosaccharide processing whereas the latter two compounds inhibit lysosomal function. The P0 synthesized by the 12-day explant cultures following the addition of swainsonine is readily fucosylated, implying that the protein has progressed at least as far as the medial Golgi before its exit and subsequent catabolism. If cultures from 4-, 6-, and 8-day-old animals are analyzed for P0 biosynthesis by [3H]mannose incorporation in the presence of swainsonine, detectable levels of the glycoprotein are seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Myelin Basic Protein in the Central Nervous System of the Jimpy and the Normal Mouse

We describe the immunohistochemical localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin basic protein (MBP) in CNS of the jimpy mutant mouse which is characterized by dys- and demyelination. In controls, the CNPase and MBP were localized exclusively in white matter in the CNS. The jimpy mutant mice were severely affected: A very weak reaction was observed in the white matter. Very few CNPase- and MBP-positive myelin sheaths were observed, and some degradation products were also observed after reaction with antisera against both CNPase and MBP. The immunohistochemical reaction in the jimpy mice showed a similar localization in both CNPase and MBP.     

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in cat peripheral nerve

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in desheathed, saline perfused cat sciatic nerve is reported. CNPase specific activity was enriched in the total particulate (P₂) fraction and was low in the soluble (S₂) fraction. “Light-myelin” floating above the 0.60 M sucrose phase had the highest CNPase activity, 2.5-fold over the crude homogenate (CH). By contrast, enzyme activity in “heavy myelin” floating above the 0.85 M sucrose interface was equal to that of the CH and accounted for only 12% of total activity. CNPase activity in the membranes floating above the 0.25 and 0.60 and 0.85 M sucrose phases comprised nearly 70% of the total CNPase activity. The “light myelin” fraction floating above the 0.60 M sucrose accounted for approx. 51% of the total CNPase activity. SDS-PAGE of membranes individually harvested from above the 0.25 and 0.60 and 0.85 M sucrose phases revealed the presence of myelin-specific proteins (P₀, P₁; and P₂). Electron microscope examination demonstrated the presence of myelin in each membrane fraction. The results of this study show that the majority of CNPase activity is associated with “light myelin” in cat peripheral nerve.     

Preparation and characterization of unilamellar myelin vesicles

Myelin vesicles have been obtained from isolated rat brain myelin and shown by electron microscopy to consist of single bilayer membranes. The yield of the preparation is approximately 25% of the myelin proteins. The vesicles show a typical myelin protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contain activity for the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase). The preparation consists of both inside-out and right-side-out vesicles, and the proportion in each orientation varies from one preparation to another. The occurrence of two populations is demonstrated by the observation that hypotonically lysed vesicles compete to a greater extent than intact vesicles in a competitive enzyme-linked immunosorbent assay with myelin basic protein antiserum. In addition, only a portion of the CNPase activity of the vesicles is trypsin-sensitive and detectable in the absence of detergent; the remaining, trypsin-insensitive activity is present in detergent-disrupted membranes. Thus, there are vesicle populations in which myelin basic protein and CNPase are accessible and others in which they are inaccessible. A population of uniformly oriented right-side-out vesicles has been obtained by ConA-Agarose affinity column chromatography and elution of the bound fraction with methyl-alpha-D-mannopyranoside. In the absence of detergent, less than 10% of the total CNPase activity of these vesicles can be demonstrated, suggesting that the active site of CNPase is opposite to that of the ConA binding site and, therefore, appears to be on the cytoplasmic face of the myelin membrane.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Oligodendrocyte-specific expression of human immunodeficiency virus type 1 Nef in transgenic mice leads to vacuolar myelopathy and alters oligodendrocyte phenotype in vitro

Vacuolar myelopathy (VM) is a frequent central nervous system complication of human immunodeficiency virus type 1 (HIV-1) infection. We report here that transgenic (Tg) mice expressing even low levels of Nef in oligodendrocytes under the regulation of the myelin basic protein (MBP) promoter (MBP/HIV(Nef)) developed VM similar to the human disease in its appearance and topography. The spinal cords of these Tg mice showed lower levels of the myelin proteins MAG and CNPase and of the 21-kDa isoform of MBP prior to the development of vacuoles. In addition, Tg oligodendrocytes in primary in vitro cultures appeared morphologically more mature but, paradoxically, exhibited a less mature phenotype based on O4, O1, CNPase, and MBP staining. In particular, mature CNPase(+) MBP(+) Tg oligodendrocytes were less numerous than non-Tg oligodendrocytes. Therefore, Nef appears to affect the proper differentiation of oligodendrocytes. These data suggest that even low levels of Nef expression in human oligodendrocytes may be responsible for the development of VM in HIV-1-infected individuals.     

Production of Monoclonal Antibody to 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase from Bovine Cerebral White Matter

Lewis rats were immunized with partially purified 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) from bovine cerebral white matter and the spleen cells were fused with cell of a mouse myeloma cell line (SP-2). The production of monoclonal antibody was detected by enzyme-linked immunoadsorbent assay, immunohistochemical staining of bovine cerebrum, Western blotting analysis, and CNPase binding assay. Monoclonal antibody that specifically binds CNPase molecules was obtained. However, the antibody did not suppress the enzyme activity. Western blotting analysis demonstrated that the monoclonal antibody binds both CNa (Wla) and CNb (Wlb). The monoclonal antibody was identified as being of the IgG2c subclass. Immunohistochemical examination revealed that the myelin sheath in the CNS was heavily stained with the monoclonal antibody in several species (bovine, mouse, rat, and human). In contrast, peripheral nervous system myelin was not stained even in bovine tissue. These results suggest that the monoclonal antibody obtained in the present study specifically recognizes the CNPase molecules in the CNS.     

Effects of Detergents, Proteins, and Lipids on 2'':3''-Cyclic-Nucleotide 3''-Phosphodiesterase Activity

The myelin marker 2':3'-cyclic-nucleotide 34'-phosphodiesterase (CNPase) was isolated to a lipid- and phosphate-free stage. The effects of exogenously added lipids were tested on this preparation and compared to the known stimulation of the enzyme by detergents and proteins. CNPase could be stimulated 2-3 fold by these various agents which appeared to be additive in their effect. Enzyme-protein and enzyme-lipid interactions and possible medical use of the improved assay conditions for CNPase employed in the study are discussed.     

Molecular cloning of the myelin specific enzyme 2',3'-cyclic-nucleotide 3'-phosphohydrolase

We describe the isolation of cDNA clones for bovine brain 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNPase, EC 3.1.4.37), the third most abundant protein in central nervous system myelin. The cDNA encodes the complete protein (400 amino acids) and hybridizes to a major size species of mRNA in bovine brain tissue, approx. 2.7 kb in size. CNPase mRNA levels do not appear to be affected in quaking dysmyelinating mutant mice. The sequence reveals probable sites for CNPase phosphorylation by cAMP-dependent protein kinase and a region of homology with haemocyanin.     

CNPase activity in myelin from adult rat brains after prolonged lead exposure in vivo

The present study investigated the sensitivity of rats cerebral myelin to prolonged toxicity of lead (Pb) that imitates environmental exposure to this metal. The results indicated that 90 days exposure of young adult rats to lead in drinking water affects the morphology of myelin sheaths, expressed in disintegration of its multilamellar structure. Both, the protein content and the activity of the myelin-specific enzyme CNPase (2',3'-cyclic nucleotide 3-phosphodiesterase), were lowered. The Michaelis-Menten kinetic for CNPase in myelin obtained from control and Pb-treated rats was different. Km increased and Vmax decreased when compared to controls. Observed disturbances in enzyme activity may be one of the potential reasons of the ultrastructural changes. It is thus tempting to speculate that Pb may be considered as a one of the factors contributing to demyelinating diseases.     

Intracellular Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in a Neuronal Cell Line as Examined by Immunofluorescence and Cell Fractionation

The enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, EC 3.1.4.37) occurs not only in myelin fractions and glial cells, but can also be shown to be present in a CNS cell line of neuronal origin (B104). Direct immunofluorescence microscopy of B104 cells with fluorescein isothiocyanate-conjugated rabbit anti-CNPase antibodies shows a discrete and specific intracytoplasmic location of CNPase. Fractionation of the cells was performed by differential centrifugation of a cell homogenate and continuous sucrose density-gradient centrifugation. As monitored by marker enzyme activities, CNPase seems to be associated with endoplasmic reticulum membranes.     

Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination

Some steroids are synthesized within the central and peripheral nervous system, mostly by glial cells. These are known as neurosteroids. In the brain, certain neurosteroids have been shown to act directly on membrane receptors for neurotransmitters. For example, progesterone inhibits the neuronal nicotinic acetylcholine receptor, whereas its 3alpha,5alpha-reduced metabolite 3alpha, 5alpha-tetrahydroprogesterone (allopregnanolone) activates the type A gamma-aminobutyric acid receptor complex. Besides these effects, neurosteroids also regulate important glial functions, such as the synthesis of myelin proteins. Thus, in cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). An important role for neurosteroids in myelin repair has been demonstrated in the rodent sciatic nerve, where progesterone and its direct precursor pregnenolone are synthesized by Schwann cells. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths.