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.     

Case-control association study of the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) gene and schizophrenia in the Han Chinese population

Converging evidence from imaging, microarray, genetic, and other studies suggests that abnormalities in myelin may play a role in schizophrenia. The expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), which is used as a myelin marker, has been reported to be reduced in the schizophrenic brain. A synonymous genetic variation in the CNP gene, rs2070106, has recently been shown to be associated with schizophrenia in Caucasians. The present study investigates whether this finding can be replicated in the Han Chinese population. We performed an association analysis of four ht-SNPs in the CNP gene in a Chinese sample consisting of 426 schizophrenic patients and 439 healthy controls. We did not find any significant differences in any genotypic, allelic or haplotypic distributions between patients and controls. Therefore, this study did not find an association between genetic variations in the CNP gene and schizophrenia in the Han Chinese population.     

A family-based association study of the myelin-associated glycoprotein and 2',3'-cyclic nucleotide 3'-phosphodiesterase genes with schizophrenia

A recent surge of evidence implicating myelin abnormalities in the etiology of schizophrenia has been found. This study is a family-based genetic association analysis examining the myelin-associated glycoprotein (MAG) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) genes in schizophrenia. About 246 families of primarily European-Caucasian origin were genotyped for MAG rs2301600, rs720308, rs720309, rs756796, and CNP rs2070106 single nucleotide polymorphisms (SNPs). The FBAT program (v1.7.2) and Transmit were used to analyze individual SNPs and haplotypes, respectively. The CNP SNP (rs2070106) was potentially associated with schizophrenia (P=0.027). MAG variants were not associated with disease transmission based on single marker or haplotype analysis. A significant maternal parent-of-origin effect for the CNP risk allele for schizophrenia was found (P=0.003). No CNP-MAG gene-gene interaction conferred increased risk for schizophrenia. Our finding provides support for potential association of the CNP gene but not the MAG gene in schizophrenia in a Caucasian population.     

Expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the amoeboid microglial cells in the developing rat brain

Expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in amoeboid microglial cells (AMC) in developing rat brain from prenatal day 18 (E18) to postnatal day 10 (P10) was demonstrated by immunohistochemistry/immunofluorescence and immunoelectron microscopy both in vivo and in vitro, respectively. Furthermore, real time-polymerase chain reaction (PCR) was performed to determine the expression of CNPase at mRNA level in cultured microglial cells in control conditions and following lipopolysaccharide stimulation. CNPase immunoreactive amoeboid microglia occurred in large numbers in the corpus callosum, subventricular zone and cavum septum pellucidum at P0 but were progressively reduced with age and were undetectable at P14. By immunoelectron microscopy, immunoreaction product was associated primarily with the plasma membrane, filopodial projections and mitochondria in AMC. Real time-PCR analysis revealed that CNPase mRNA was expressed by cultured amoeboid microglia and was significantly up-regulated in microglial activation induced in vitro by lipopolysaccharide. The functional role of CNPase in AMC remains speculative. Given its expression in AMC transiently occurring in the perinatal brain and that it is markedly elevated in activated microglia, it is suggested that the enzyme may be linked to the major functions of the cell type such as release of chemokines and cytokines. In relation to this, CNPase may play a key role associated with transportation of cytoplasmic materials.     

Encephalitogenicity of myelin-associated oligodendrocytic basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase for BALB/c and SJL mice.

In search of new encephalitogenic myelin antigens, the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and 19 000 MW isoform of myelin-associated oligodendrocytic basic protein (MOBP) were obtained as recombinant proteins by the baculovirus expression system in Spodoptera frugiperda cells and purified to homogeneity by immobilized metal chelate affinity chromatography (IMAC). The purified MOBP was soluble in water and showed retarded migration on sodium dodecyl sulphate-polyacrylamide gel electrophoresis similar to myelin basic protein (MBP). MOBP induced experimental autoimmune encephalomyelitis (EAE) in nine of 15 susceptible SJL OlaHsd mice, causing death in two animals, whereas three of 14 BALB/c mice showed mild symptoms of EAE, manifested as transient weakness of hind limbs. In both mouse strains, periventricular infiltrates of mononuclear cells were observed. In addition, both 46 000 MW and 48 000 MW CNP isoforms were shown to be non-encephalitogenic for both mouse strains.     

Expression of the non-compact myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in olfactory bulb ensheathing glia from explant cultures

The non-compact myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) may have a unique role in signaling pathways mediated by lipid-protein domains (Kim and Pfeiffer, 1999). We have tested for CNPase in olfactory bulb ensheathing glia (OBEG) in explant cultures. Migrating bipolar and multipolar cells with OBEG typical morphologies were inimunoreactive for both vimentin- and S100-like proteins. Although apparently devoid of myelin basic protein (MBP)-like immunoreactivity, these cells displayed weak but unambiguous CNPase-like immunoreactivity. Our results suggest a further resemblance to myelinating Schwann cells.     

Decreased brain levels of 2',3'-cyclic nucleotide-3'-phosphodiesterase in Down syndrome and Alzheimer's disease.

In Down syndrome (DS) as well as in Alzheimer's disease (AD) oligodendroglial and myelin alterations have been reported. 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and carbonic anhydrase II (CA II) are widely accepted as markers for oligodendroglia and myelin. However, only data on CNPase activity have been available in AD and DS brains so far. In our study we determined the protein levels of CNPase and CA II in DS, AD and in control post mortem brain samples in order to assess oligodendroglia and myelin alterations in both diseases. We used two dimensional electrophoresis to separate brain proteins that were subsequently identified by matrix assisted laser desorption and ionization mass-spectroscopy (MALDI-MS). Seven brain areas were investigated (frontal, temporal, occipital and parietal cortex, cerebellum, thalamus and caudate nucleus). In comparison to control brains we detected significantly decreased CNPase protein levels in frontal and temporal cortex of DS patients. The level of CA II protein in DS was unchanged in comparison to controls. In AD brains levels of CNPase were decreased in frontal cortex only. The level of CA II in all brain areas in AD group was comparable to controls. Changes of CNPase protein levels in DS and AD are in agreement with the previous finding of decreased CNPase activity in DS and AD brain. They probably reflect decreased oligodendroglial density and/or reduced myelination. These can be secondary to disturbances in axon/oligodendroglial communication due to neuronal loss present in both diseases. Alternatively, reduced CNPase levels in DS brains may be caused by impairment of glucose metabolism and/or alterations of thyroid functions.     

Immunohistochemical localization of myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase in flattened membrane expansions produced by cultured oligodendrocytes

Oligodendrocytes, the cells responsible for myelin sheath formation in the central nervous system, were isolated from primary dissociated mixed glial cultures prepared from newborn mouse forebrain, and further cultured in a serum-free defined culture medium. Single and double indirect immunofluorescence using antibodies against the myelin glycolipids, galactocerebroside and sulfatide, and the myelin proteins, myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase, was used to investigate the composition of the flat membrane extensions produced by some oligodendrocytes in culture. Galactocerebroside and sulfatide were both expressed on the external surface of the plasma membrane of oligodendrocyte cell bodies and processes and also the membrane expansions. Neither myelin basic protein nor 2',3'-cyclic nucleotide 3'-phosphohydrolase were expressed on the external surface of oligodendrocytes. Myelin basic protein could be localized to the cell body and the membrane expansions but not the major and fine processes. The localization of these myelin components suggests that the expansions have characteristics of the mature myelin membrane. 2',3'-Cyclic nucleotide 3'-phosphohydrolase was found to be localized in the cell body, and in total contrast to myelin basic protein, in the major processes and the fine interconnecting processes, but not the membrane expansions. In some of the cells 2',3'-cyclic nucleotide 3'-phosphohydrolase was present at the outer extremities of the flat membrane sheets, giving the appearance of an extending growth region. Our results thus clearly show that 2',3'-cyclic nucleotide 3'-phosphohydrolase is localized within oligodendrocytes in discrete regions of plasma membranes and suggest that this protein has a possible role in the early stages of myelin formation.     

2',3'-cyclic nucleotide-3'-phosphodiesterase activity as an index of myelin in the post-mortem brains of patients with Alzheimer's disease

The activity of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) was determined as an index of myelin in the post-mortem brain samples of 16 patients with Alzheimer's disease (AD) and of 14 controls. The CNPase activity was mildly increased in the temporal, hippocampal and parietal cortex in AD subjects pointing to a relative sparing of myelin as compared to the neuron loss within cortex in AD. In the hippocampus the CNPase activity was decreased in AD patients indicating loss of myelin and thus myelinated axons in this area in AD.     

Arylsulphatase A and 2',3'-cyclic nucleotide 3'-phosphohydrolase activities in the brains of myelin deficient mutant mice

Arylsulphatase A and 2',3'-cyclic nucleotide 3'-phosphohydrolase activities of myelin deficient mutant mice brains were studied. The results indicated that there were no changes in arylsulphatase A activity of the developing mutant brain, whereas the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase decreased considerably. The data obtained in this study suggest that in brain arylsulphatase A activity is localized in cells other than oligodendroglia.     

Relationship of 2',3'-cyclic nucleotide 3'-phosphohydrolase activity of large enveloped RNA viruses to host cell activity.

Purified virions of the large RNA viruses show 2',3'-cyclic nucleotide 3'-phosphohydrolase (3'-CNPase) activity. The 3'-CNPase activity is virion-associated and stimulated by their treatment with nonionic detergents. Cytopathic viruses such as influenza A2 (Singapore/57), NDV, and VSV showed the specific activity of a virion-associated 3'-CNPase equal to or lower than the specific activity of host cell enzyme. Retroviruses are an example of extreme relationship of 3'-CNPase to virion. With the AMV-BAI-A associated enzyme activity increased after Triton X-100 treatment ten times more than that associated with other viruses examined. The specific activity of virus-associated 3'-CNPase was 16-28 times higher than that in chick myeloblasts. BLV showed a very low enzyme activity. The correlation between the activity of cellular 3'-CNPase and virus yield showed that 3'-CNPase could belong to cellular factors influencing virus replication.     

A histochemical method for demonstration of 2',3'-cyclic 3'-phosphohydrolase activity

A histochemical method for demonstration of 2',3'-cyclic nucleotide 3'-phosphohydrolase activity is proposed. Cryostat sections, fixed in chlorophorm-methanol mixture, are incubated in a solution containing cyclic adenosine 2',3'-monophosphate, acid phosphatase, lead nitrate in acetate buffer, pH = 6,4 or 6.5. This method is based on conversion by the 2',3'-cyclic nucleotide 3'-phosphohydrolase of cyclic adenosine 2',3'-monophosphate to nucleotide-2'-monophosphate. Under the action of exogenous acid phosphatase, inorganic phosphate is released. It precipitates as lead phosphate if lead ions are present. By adding yellow ammonium sulphide, the lead phosphate is converted to lead sulphide which is the visible reaction product.     

Hydrolysis of ribonucleoside 2',3'-cyclic phosphates by influenza and Newcastle disease viruses

Two enzymatic activities hydrolysing ribonucleoside 2', 3'-cyclic phosphates (2', 3'-cNMP) to 2'- or 3'- nucleoside monophosphate were found associated with influenza and Newcastle disease viruses. The two enzymatic activities differed from each other by temperature optima and thermoresistance. 2', 3'-Cyclic nucleotide 3'-phosphohydrolase was responsible for splitting of the substrate to 2'-NMP. Splitting of the substrate to 3'-NMP was due either to ribonuclease or to 2', 3'-cyclic nucleotide 2'-phosphohydrolase.     

ERV3, a full-length human endogenous provirus: chromosomal localization and evolutionary relationships

A full-length human endogenous provirus termed ERV3 was isolated from a human fetal recombinant DNA library by low stringency hybridization with two probes: baboon endogenous virus LTR; and a pol-env subclone from the endogenous chimpanzee provirus, CH2. DNA sequencing within the clone and comparisons with other retroviruses revealed that ERV3 contains gag and pol gene sequences that are significantly related to those of mammalian type C retroviruses and previously described human endogenous proviruses. The ERV3 genome was determined to reside at a single locus on human chromosome 7 using a panel of rodent X human somatic cell hybrids.     

Cloning, experession and chromosomal localization of a member of the human cytochrome P450IIC gene sub-family

We have isolatd and sequenced a cDNA clone (pB8) that codes for a novel member of the dcytochrome P450IIC sub-family of man. Analysis, by Southern blot hybridization, of DNA isolated from a panel of nine independent human-rodent somatic cell hybrids demonstrated that the corresponding gene ( CYP2C ) is located on human chromosome 10. Northern blot hybridization of RNA isolated from human livers revealed a 10-fold inter-indicidual variation in the expression of the gene.     

Elevated arteriolar adenosine 3',5'-cyclic monophosphate production by SHR

Adenosine 3',5'-cyclic monophosphate (cAMP) metabolism was studied in the microcirculation (100- to 150-micrometers arterioles) of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) at different stages of hypertension. Mesenteric arterioles from animals 4, 6, 12, and 18 wk old were incubated in Krebs-Ringer bicarbonate buffer for 30 min at 37 degrees C, pH 7.4, with and without the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX). cAMP was assayed by radioimmunoassay. Arteriolar production of cAMP was age related in both WKY and SHR rats although the temporal patterns were different. At 6 wk (developmental stage of hypertension in SHR) cAMP accumulation in the presence or absence of MIX by SHR arterioles was higher than in the WKY before falling to normotensive levels at 12 wk. Salbutamol (a beta 2-agonist) stimulated dose-dependent increases in cAMP in both WKY and SHR at 6 wk. Stimulation of cAMP by salbutamol or by isoproterenol was blocked by propranolol. Neither agonist increased guanosine 3',5'-cyclic monophosphate. These data indicate that differences in cAMP metabolism are evident at the arteriolar level during the developmental stage of SHR hypertension. These differences may contribute to the morphological and physiological changes occurring at this time.