Expression of the oligodendrocyte marker 2'3'-cyclic nucleotide 3'-phosphodiesterase in non-glial cells

The 46 kD isoform of the 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPI) was expressed in HeLa cells by transfection of its cDNA clone. The distribution of this polypeptide as mapped by indirect immunofluorescence and conventional epifluorescence microscopy appeared diffuse and generally uniform throughout the cytoplasm. Confocal microscopic imaging and analysis of pseudocolored images confirmed this distribution but also revealed that there was a high concentration of CNPI near the plasma membrane of the cell. This pattern is very similar to that observed by immunoelectronmicroscopy of myelinating oligodendrocytes (Trapp et al.: J Neurochem 51:859-868, 1988; Braun et al.: J Neurosci 8: 3057-3066, 1988). These results suggest that CNP may interact with a membrane-associated molecule that is not unique to oligodendrocytes.     

Convergent evidence for 2',3'-cyclic nucleotide 3'-phosphodiesterase as a possible susceptibility gene for schizophrenia

CONTEXT: Convergent data make 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) a candidate gene for schizophrenia. Reduced expression has been reported in the schizophrenic brain. The CNP gene maps to a region to which we have reported linkage to schizophrenia. Mice in which the CNP gene has been knocked out display central nervous system pathological characteristics reminiscent of some features observed in schizophrenia. 2',3'-Cyclic nucleotide 3'-phosphodiesterase is used as a marker of myelin-forming cells and is detectable in cells of oligodendrocyte lineage throughout life. Because CNP is thought to be important for oligodendrocyte function, altered expression has been interpreted as supportive of the hypothesis that altered oligodendrocyte function may be an etiological factor in schizophrenia. However, it is unclear whether the observed changes in the schizophrenic brain are primary or secondary. OBJECTIVES: To determine if CNP expression is influenced by DNA polymorphisms and to verify if these polymorphisms are associated with schizophrenia. DESIGN: Allele-specific messenger RNA expression assay and genetic association studies. SETTING: Unrelated subjects were ascertained from secondary psychiatric inpatient and outpatient services. PARTICIPANTS: We used brain tissue from 60 anonymous individuals with no known psychiatric disorder; a case-control sample of 708 white individuals from the United Kingdom meeting DSM-IV criteria for schizophrenia matched for age, sex, and ethnicity to 711 blood donor controls; and a pedigree with DNA from 6 affected siblings and 1 parent, showing evidence for linkage to CNP. MAIN OUTCOME MEASURES: Association between allele and gene expression. Association between allele and schizophrenia. RESULTS: The exonic single nucleotide polymorphism rs2070106 was associated with CNP expression (P<.001). Compatible with underexpression of CNP messenger RNA in schizophrenia, the lower-expressing A allele was significantly associated with schizophrenia (P = .04) in the case-control sample. All affected individuals in the linked pedigree were homozygous for the lower-expression allele, providing independent support for the association (P = .03). CONCLUSIONS: Our data support the hypothesis that reduced CNP expression in the schizophrenic brain is relevant to disease etiology and therefore provide support for the general hypothesis that altered oligodendrocyte function is an etiological factor in schizophrenia.     

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.     

Monoclonal antibody Rip specifically recognizes 2',3'-cyclic nucleotide 3'-phosphodiesterase in oligodendrocytes

The antigen recognized with monoclonal antibody (mAb) Rip (Rip-antigen) has been long used as a marker of oligodendrocytes and myelin sheaths. However, the identity of Rip-antigen has yet to be elucidated. We herein identified the Rip-antigen. No signal recognized by mAb-Rip was detected by immunoblot analyses in the rat brain, cultured rat oligodendrocytes, or the oligodendrocyte cell line CG-4. As this antibody worked very well on immunocytochemistry and immunohistochemistry, Rip-antigen was immunopurified with mAb-Rip from the differentiated CG-4 cells. Eight strong-intensity bands thus appeared on 5-20% SDS-PAGE with SYPRO ruby fluorescence staining. To identify these molecules, each band extracted from the gel was analyzed by MALDI-QIT/TOF mass spectrometry. We found an interesting molecule in the oligodendrocytes from an approximately 44-kDa band as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). To test whether CNP was recognized by mAb-Rip, double-immunofluorescence staining was performed by using Alexa Fluor 488-conjugated mAb-Rip and Alexa Fluor 568-conjugated mAb-CNP in the rat cerebellum, mouse cerebellum, cultured rat oligodendrocytes, and CG-4 cells. The Rip-antigen was colocalized with CNP in these cells and tissues. To provide direct evidence that CNP was recognized by mAb-Rip, rat Cnp1-transfected HEK293T cells were used for double-immunofluorescence staining with mAb-Rip and mAb-CNP. The Rip-antigen was colocalized with CNP in rat Cnp1-transfected HEK293T cells, but the antigen was not detected by mAb-Rip and mAb-CNP in mock-transfected HEK293T cells. Overall, we have demonstrated that the antigen labeled with mAb-Rip is CNP in the oligodendrocytes.     

Age-dependent accumulation of ubiquitinated 2',3'-cyclic nucleotide 3'-phosphodiesterase in myelin lipid rafts

Changes in brain white matter are prominent features of the aging brain and include glial cell activation, disruption of myelin membranes with resultant reorganization of the molecular components of the node of Ranvier, and loss of myelinated fibers associated with inflammation and oxidative stress. In previous studies, overexpression of CNP, a key myelin protein, was implicated in age-related changes in myelin and axons. Here we examine the extent of CNP accumulation in brain white matter and isolated myelin of aged rhesus monkeys and its relationship to CNP degradation and partitioning in myelin. With age, excess CNP is found in myelin and throughout brain white matter accompanied by proteolytic fragments of CNP. These increases occur in the absence of changes in CNP mRNA levels. Using a combination of 2D electrophoresis, immunoprecipitation, and mass spectrometry analysis, ubiquitinated CNP was demonstrable in the Triton X-100 insoluble lipid raft associated fractions of myelin isolated from rhesus monkeys. Further, using ubiquitin-mediated fluorescence complementation (UbFC), ubiquitinated CNP was visualized by microscopy in both COS-7 and MO3.13 cells and by immunoblot in MO3.13 cells and appears to at least partially localize within lipid rafts. The findings suggest that incomplete degradation of CNP due to failure of the proteasomal system and aberrant degradation by calpain-1 leads to age-related CNP accumulation and proteolysis. In sum, we suspect these phenomena result in age-related dysfunction of CNP in the lipid raft, which may lead to myelin and axonal pathology.     

T cell response to 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in multiple sclerosis patients

T cell responses targeting myelin antigens are possibly involved in the pathogenesis of demyelinating diseases, such as multiple sclerosis (MS). Little is known about human T cell responses to 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), the third most abundant myelin protein. We examined the primary peripheral T cell response to CNPase and characterized CNPase-specific CD4+ long-term T cell lines (TCL) from MS patients and healthy donors. The strongest primary responses were found in two MS patients with very active disease and were directed against CNP(343-373). We identified immunodominant epitope clusters in the regions CNP(343-373) and (356-388) that were recognized in the context of MS-associated HLA-DR2 and DR4 molecules. These data provide the immunological basis for further investigation of CNPase as a potential target self-antigen in MS.     

Gene expression of the central and peripheral nervous system myelin membrane 2', 3'-cyclic nucleotide 3'-phosphodiesterase in development

Determination of gene expression for the 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) during development shows that the amount of translatable RNA encoding this enzyme increases in parallel with myelination in the mouse brain. The two isoforms of this enzyme are developmentally regulated in parallel, in contrast to several other myelin proteins. By comparison, the amount of translatable RNA encoding CNP in the peripheral nervous system (rat sciatic nerve) remains constant during development. The two isoforms that are expressed are identical in apparent molecular weight to rat brain CNP. Moreover, no apparent differences were detected by proteolytic digestion (Cleveland mapping) of CNP 1 from the central or peripheral nervous systems.     

Expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in the developing olfactory bulb and subventricular zone rostral extension

The olfactory bulb (OB) presents a unique pattern of permanent acquisition of primary afferents and interneurons, but not much detail is known about the differentiation of its oligodendroglial cells. We studied the expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a protein related to axonal ensheathment by myelinating cells. Expression of CNPase in OB follows a general caudorostral gradient, with the exception of the glomerular layer (GL). At postnatal day 5-6 (P5-P6), the first CNPase(+) profiles appeared in the dorsal lateral olfactory tract adjacent to the accessory OB (AOB), followed by rare cell bodies and processes in AOB internal plexiform layer at P7. At P9, the main OB (MOB) granular cell layer (GrCL) already showed intensely stained CNPase(+) processes. From P5 to P12, small numbers of CNPase(+) cells were found in the subventricular zone (SVZ), throughout its rostral extension (SVZ-RE), and in the intrabulbar subependymal layer. The appearance of CNPase(+) profiles delimiting glomeruli started in the GL rostralmost region at P12, extending to all GL levels, but glomeruli remained open caudally at P15. At P18, oligodendroglial glomeruli were evident throughout OB, but the adult pattern was established only after P30. There was no age-related loss of CNPase immunoreactivity in glial cell bodies, possibly indicating de novo ensheathment of neurites. Our results show an earlier onset of oligodendroglial differentiation in OB than previously reported and a rostrocaudal gradient of formation of oligodendroglial glomeruli. They also raise the possibility that a minor fraction of OB oligodendrocytes might derive from the SVZ-RE.     

Chromosomal locations of genes encoding 2',3' cyclic nucleotide 3'-phosphodiesterase and glial fibrillary acidic protein in the mouse

Cyclic nucleotide phosphodiesterase (CNP) and glial fibrillary acidic protein (GFAP) are useful markers of myelin and astroglia, respectively. Two proteins with CNP activity are known to exist in brain and lymphoid tissues. They appear to be the products of several distinct but related messenger ribonucleic acid (mRNA) species. GFAP is a single protein encoded by a single mRNA. We have localized the GFAP gene to distal chromosome 11 in the mouse. There are two genetic loci identified by CNP probes, one is closely linked to the GFAP gene, and the other maps to chromosome 3.     

Ethanol exerts different effects on myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase expression in differentiating CG-4 oligodendrocytes

Evidence suggests that abnormal myelination is one factor contributing to the neuoropathology associated with fetal alcohol syndrome. We investigated the potential teratogenic effects of ethanol (EtOH) on myelin formation by determining its effects on the developmentally regulated increased expression of myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in differentiating CG-4 oligodendrocytes (OLGs). By using CG-4 OLGs in vitro we identified processes altered by ethanol actions exerted directly on OLGs. During the first 8 days of development, EtOH decreased the expression of the major structural 18.5 and 14 kDa MBP isoforms by at least 40% at 4 days of development. EtOH concentrations between 25 and 75 mM inhibited MBP expression in a dose-dependent manner. Adding or withdrawing EtOH on specific days of differentiation reversibly modulated the expression of MBP, and the degree of inhibition was directly related to the length of ethanol exposure. As little as two consecutive days of EtOH exposure either early or late during development caused at least a 20% inhibition, however, no short critical time window of EtOH vulnerability for the inhibition was observed. The ethanol effect was selective for MBP expression, as EtOH did not alter the developmentally-regulated increased expression of CNP isozymes or enzyme activity. The results indicate that one factor contributing to the development of fetal alcohol syndrome may be defective myelination resulting from delayed and decreased MBP expression.     

Immunocytochemical localization by electron microscopy of 2'3'-cyclic nucleotide 3'-phosphodiesterase in developing oligodendrocytes of normal and mutant brain

Oligodendrocytes or their putative progenitors were the only cells found to be immunoreactive to polyclonal antisera against the enzyme 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in developing and mature brains of rats and mice, as visualized by light and electron microscopy. Prior to myelination (day 6), oligodendrocytes of the corpus callosum have reticular networks of CNP-containing filopodia, in addition to abundant CNP throughout the cytoplasm. Some glioblast-like cells of the subventricular zone are also immunoreactive to anti-CNP, suggesting that, as progenitors of oligodendroglia, they express this myelination-related protein as one of the earliest events in myelinogenesis. Following the commencement of myelination (day 15), many oligodendrocytes lose much of their lacelike network of fine projections, possessing, instead, larger CNP-filled processes that extend to myelin-bearing fibers. CNP was always found only in the cytoplasm-containing compartments of the cells and myelin sheaths; neither lamellae nor cellular membranes were immunostained. These data support our contention that CNP is not an intrinsic membrane protein, despite its strong interaction with membrane components when cells are disrupted. In mutant (mld) mice (day 25), the many distended and uncompacted oligodendroglial processes that invest axons with only a few turns of membrane contained cytoplasmic CNP, accounting for the elevated levels of CNP activity previously noted in tissue fractions.     

Temporal expression of myelin-specific components in neonatal mouse brain cultures: evidence that 2',3'-cyclic nucleotide 3'-phosphodiesterase appears prior to galactocerebroside

A range of antibodies to cell-specific markers was employed to characterize and quantify the cell types present in neonatal mouse brain cell cultures. Astrocytes were the major cell type to develop in culture and formed a bed layer by 5-7 days in vitro (DIV). A population of cells appeared on this layer which consisted of oligodendrocytes, progenitor cells and microglia. Some neurons were detected in the cultures up to at least 27 DIV. With time, the number of progenitor cells decreased in the cultures and there was a concomitant increase in the number of oligodendrocytes. Maximal numbers of cells expressing galactocerebroside (GC), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin-associated glycoprotein were observed at 18 DIV. The highest number of cells expressing myelin basic protein and proteolipid protein were observed at 25-30 and 35-40 DIV, respectively. Double-label studies with antibodies to A2B5/CNP and A2B5/GC showed that several A2B5+,CNP+ cells were present at 6 DIV. In contrast, no A2B5+,GC+ cells could be seen at this age. Confirming these findings, some CNP+,GC- cells were observed when cells were double-labeled with antibodies to CNP and GC. These findings suggested that the expression of CNP precedes that of GC in oligodendrocytes in these cultures.     

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.     

Activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in human cerebrospinal fluid.

Activity of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was assayed in cerebrospinal fluid samples obtained from patients with multiple sclerosis (MS) and other neurological diseases. The enzyme activity was found to be elevated in acute cases of MS and reduced during remission. It was present in other demyelinating diseases, and no activity was detected in normal CSF. CNP may be released into CSF from any insult to myelin. The level of activity appears to reflect demyelination and the rate of breakdown of the myelin sheath.     

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.