Immunochemical characterization of antibodies to the myelin proteolipid protein (PLP)

The immunochemical specificity of antibodies raised against the bovine myelin proteolipid protein (PLP) and a series of PLP synthetic peptides was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. Polyclonal rabbit anti-bovine PLP antibodies were cross-reactive with PLP isolated from rat, monkey, and human CNS white matter, as well as with PLP incorporated into rat myelin vesicles. Immunochemical analyses of 11 anti-peptide antisera revealed a more restrictive cross-reactivity pattern with only five of the 11 antisera, against peptides encompassing residues 48-59, 97-105, 183-193, 192-200 and 264-276, cross-reacting with bovine PLP by ELISA. Immunoblot analyses with the anti-peptide antisera demonstrated a similar recognition with four of the 11 antisera against peptides 48-59, 97-105, 192-200 and 264-276 recognizing the myelin PLP. These data suggest that, despite its strong phylogenetic conservation, multiple antigenic sites exist within the PLP molecule, and some of these determinants can be mimicked by synthetic peptides. However, the restrictive cross-reactivities of the anti-peptide antisera suggest that humoral recognition of the myelin PLP is conformationally dependent. The availability of anti-peptide antibodies of predetermined specificity, capable of recognizing the intact protein, should permit detailed examination of PLP topography in various experimental systems.     

Cyclic AMP-induced upregulation of proteolipid protein and myelin associated glycoprotein gene expression in C6 cells.

A model culture system of C6 rat glioma cells was used to test the involvement of cAMP in the regulation of the myelin PLP and MAG genes. The treatment of cells with isoproterenol (10(-5) to 10(-8) M) upregulated the expression of the PLP and MAG genes in a concentration-dependent manner. The mRNA for PLP reached a maximum (sevenfold higher than in control cells) after about 12-24 hr, then declined to approximately fourfold over the control level. The response of MAG gene was delayed by at least 36 hr, and the level of MAG mRNA reached a maximum of approximately 48-fold over the control level on the fourth day in culture. The co-administration of propranolol blocked the effect of isoproterenol, whereas 10(-5) M forskolin simulated the effect of isoproterenol, indicating a role of cAMP in the signal transduction cascades leading to upregulation of the myelin genes. However, the dissimilarity in the timing and the extent of upregulation of the PLP and MAG genes by cAMP-stimulating agents indicate the existence of different intracellular mechanisms for the activation of these two genes. Cycloheximide blocked the stimulatory effect of isoproterenol on both the PLP and MAG genes, indicating that the effect of cAMP on the myelin genes is mediated by protein product(s) of other cAMP-response gene(s).     

Electroblot analysis of the myelin proteolipid protein

The myelin proteolipid has been studied by the electroblot method of Towbin et al [1979]. Samples were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with proteolipid antibody. The proteolipid band could be identified immunologically in CNS myelin and in whole brain homogenates. No proteolipid was detected in PNS myelin. Proteolipid from human, bovine, rat, and mouse myelin all cross-react when analyzed by this method. No cross-reactivity was indicated between proteolipid and myelin basic protein.     

Lipophilin (PLP) gene in X-linked myelin disorders

There are several X-linked diseases in animals and at least one in man in which there is a failure of CNS myelination. We have recently cloned cDNAs for lipophilin (PLP) with which PLP sequences were localized to a region of the long arm of the X chromosome (Xq13-q22 in man) close to the jimpy (jp) locus in that mouse mutant. The present communication pursues the postulate that some of this class of diseases may involve mutations at the PLP locus. Blot hybridization analysis of PLP mRNA levels revealed a five-to tenfold reduction in the brains of hemizygous jp/Y mice. The major PLP mRNA species of those mice was also reduced in size. However, Southern blots of jp DNA digested with many different restriction enzymes failed to detect major deletions or other rearrangements in the PLP gene. A human PLP cDNA was isolated and employed to similarly analyze DNA from four patients diagnosed as having Pelizaeus-Merzbacher disease. In one of these four a significant rearrangement of the PLP gene was found. These findings suggest that there may be alterations in the PLP gene in both jp mouse and Pelizaeus-Merzbacher disease.     

Rumpshaker-like proteolipid protein (PLP) ratio in a mouse model with unperturbed structural and functional integrity of the myelin sheath and axons in the central nervous system.

The gene plp on the X chromosome encodes the isoforms proteolipid protein (PLP) and DM(20), two dominant integral membrane proteins of central nervous system (CNS) myelin. DM(20) results from the activation of the cryptic splice site in exon III of the PLP gene. We inserted a sense-orientated loxP flanked neomycin-gene into intron III of the plp sequence, using homologous recombination in embryonic stem cells and generated the homozygous neoS mouse line. Unlike the previously described complete PLP/DM(20) ablation (plp(-/-)), which has been obtained by introducing a neo-gene in antisense-orientation in the same position of intron III, the plp expression surprisingly revealed reduced mRNA levels. The PLP isoform was reduced to 50%, but DM(20) expression was unaffected. This protein pattern resembles the expression profile of the PLP isoforms in the natural occurring rumpshaker mutant. Electron microscopic examination revealed a normal compaction of CNS-myelin and maintenance of axon integrity. PLP expression levels of the wt control were recovered by Cre excision of the neo-selection gene after intercrossing neoS mice and oligodendrocyte-specific Cre-mice. These data strongly hint at different functions of intron III in PLP/DM(20)-specific splicing and mRNA stability. Furthermore evidence is provided for functionally affected translation products of the PLP gene in the rumpshaker mutant, whereas no PLP-isoform occur in plp(-/-) mice generated by introducing a selectable marker into intron III in antisense orientation.     

Myelin structure in proteolipid protein (PLP)-null mouse spinal cord

Fixed preparations of proteolipid protein (PLP)-null mouse spinal cord show myelin sheaths which in some regions consist of typical alternating major dense lines (MDLs) and intermediate lines (ILs) with a repeat period of 10.3 nm. More commonly, the lamellar structure consists of what appears to be a single population of dense lines, having a repeat period of 5.2 nm. These apparently equivalent lines are, however, sometimes distinguishable as MDLs or ILs based on continuity with cytoplasmic or extracellular regions. Focal separations of lamellae at the intermediate line are common. MDLs too may be replaced focally by cytoplasmic pockets, sometimes in the same quadrant over several lamellae, resembling Schmidt-Lanterman clefts. Occasional densities reminiscent of the “radial component” can be seen. Otherwise, this structure, which is prominent in wild-type myelin, is conspicuously absent. Redundant folding of some lamellae but not others may occur in the same sheath. These observations conform to those made previously on the isolated myelin segments that occur in the myelin-deficient rat central nervous system (CNS), which also lacks PLP. Thus, a compact lamellar structure can be seen in fixed PLP-null myelin, but defects in the apposition of both the extracellular and the cytoplasmic surfaces of the myelin membranes are common. The abnormalities seen suggest a lack of firm intermembrane bonding, resulting in structural instability. PLP-null myelin may therefore be more susceptible than normal myelin to disruption by mechanical or osmotic stresses. Although PLP is not essential for the formation of either major dense lines or intermediate lines, it may play a role in stabilizing the compact structure. © 1996 Wiley-Liss, Inc.     

Structure and expression of the mouse myelin proteolipid protein gene

The gene for the mouse myelin proteolipid protein has been isolated and the seven exons have been sequenced. Since the sequence of a rat proteolipid protein cDNA and partial sequence of the human proteolipid protein gene have been determined, it was possible to demonstrate a very high degree of conservation for the proteolipid protein gene exons among species. While there are some nucleotide changes, the protein coding region of the mouse gene encodes protein that is totally conserved relative to both rat and human proteolipid proteins. The regulatory and noncoding regions of the proteolipid protein gene are also highly conserved. The upstream regulatory and 5'-noncoding region of the gene is 92% homologous to the comparable region of the human proteolipid protein gene, and the 3'-noncoding region of the mouse gene is approximately 90% homologous to a rat proteolipid protein cDNA through 2,200 nucleotides of 3'-noncoding DNA. S1 nuclease protection experiments indicated that the major 5'-end for proteolipid protein mRNAs from mouse, rat, human, or baboon is approximately 147-160 nucleotides upstream from the initial methionine codon of the protein coding region. Other S1 nuclease protection experiments indicated the possible existence of an alternative splice site within exon 3, which may produce mRNA for DM20. This mRNA is approximately 100 nucleotides shorter than that for the proteolipid protein, and it is missing the latter half of exon 3, that is, amino acids 116-150 of the proteolipid protein sequence.     

Expression of the myelin proteolipid protein gene in the human fetal thymus

We have analyzed human fetal thymus and spleen for expression of the proteolipid protein (PLP) gene. We demonstrate that the PLP gene is transcribed in both tissues, and that both the PLP and DM-20 mRNAs are produced. Western blot analyses revealed that both the PLP and DM-20 protein isoforms were present in the fetal thymus and spleen. Immunohistochemical analyses indicated that the PLP/ DM-20 proteins were detected in cells which have the distribution and morphology of thymic macrophages. These results provide further evidence that the PLP and DM-20 proteins are expressed in cell types other than myelin forming cells and possess function(s) unrelated to myelin structure. Furthermore, these data demonstrate that the PLP and DM-20 proteins are not shielded from the immune system behind the blood-brain barrier. These observations directly impinge upon the debate concerning acquisition of tolerance and the recognition that the encephalitogenic nature of PLP in diseases, such as Multiple Sclerosis, may not simply be related to its ‘sequestration’ from a ‘naive’ immune system.     

Characterization of an intronic enhancer that regulates myelin proteolipid protein (Plp) gene expression in oligodendrocytes

The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein (approximately 50%) present in mature myelin from the central nervous system (CNS). Plp gene activity is low to nonexistent early in development but sharply increases, concurrently with the active myelination period of CNS development. Work from our laboratory suggests that the temporal regulation of Plp gene expression in mice is mediated by a positive regulatory element located within Plp intron 1 DNA. We have termed this regulatory element/region ASE (for antisilencer/enhancer). The ASE is situated approximately 1 kb downstream of exon 1 DNA and encompasses nearly 100 bp. To understand the mechanisms by which the ASE augments Plp gene expression in oligodendrocytes, Plp-lacZ constructs were generated and transfected into a mouse oligodendroglial cell line (N20.1). Results presented here demonstrate that upstream regulatory elements in the Plp promoter/5'-flanking DNA are not required for ASE activity; the ASE worked perfectly well when the thymidine kinase (TK) promoter was substituted for the Plp promoter. However, the relative location of the ASE appears to be important. When placed upstream of 2.4 kb of Plp 5'-flanking DNA, or downstream of the lacZ expression cassette, the ASE was no longer effective. Thus, the ASE might have to be in the context of the intron in order to function. To begin to identify the crucial nucleotides within the ASE, orthologous sequences from rat, human, cow, and pig Plp genes were swapped for the mouse sequence. Results presented here demonstrate that the orthologous sequence from rat can substitute for the mouse ASE, unlike those from human, cow, or pig.     

Expression of myelin proteolipid and basic protein mRNAs in cultured cells

Studies were undertaken to investigate the regulation of myelin-specific mRNA expression in cultured cells. Three experimental systems were investigated: primary oligodendrocytes grown as enriched cell populations, primary oligodendrocytes grown in the presence of chick spinal cord neurons, and C6 cells. cDNA probes specific for the myelin proteolipid mRNA and the myelin basic protein mRNA were used to quantitate proteolipid and myelin basic protein mRNA levels in cells under different experimental conditions. C6 cells expressed less than 0.2% of the proteolipid mRNA that was expressed in primary oligodendrocytes. Primary oligodendrocytes expressed the myelin-specific mRNAs for at least 104 days in culture, and the level of these mRNAs in cultures was elevated fourfold by coculturing rat oligodendrocytes with chick spinal cord neurons.     

Orientation of the myelin proteolipid protein C-terminus in oligodendroglial membranes.

The topology of the integral membrane proteolipid protein (PLP) has important structural and functional implications for central nervous system myelin. To determine the orientation of the carboxyl-terminal portion of PLP, cultured mouse oligodendrocytes were probed with polyclonal antibodies raised against a synthetic terminal peptide corresponding to PLP residues 264-276 and with ten separate monoclonal antibodies that react with this region. Cells were examined by double-label indirect immunofluorescence for the presence of the PLP C-terminus and either oligodendrocyte-specific surface or intracellular antigens. To detect surface antigens, both living and paraformaldehyde-fixed cells were incubated with primary antibodies and then stained with fluorochrome-conjugated second antibodies. Antigens located within the cytoplasmic space were identified after fixation and permeabilization of cells. Live-labeled oligodendrocytes were stained brightly for myelin-oligodendrocyte glycoprotein, galactocerebroside, and other surface markers but did not stain for the PLP C-terminus or the intracellular proteins myelin basic protein and beta-tubulin. Fixation alone was sufficient for partial permeabilization of oligodendrocytes to antibodies and resulted in limited staining of the PLP C-terminus and intracellular proteins. The permeabilized oligodendrocytes stained intensely for the PLP C-terminus, myelin basic protein, and beta-tubulin. Finally, trypsinization of living oligodendrocytes eliminated surface myelin-oligodendrocyte glycoprotein staining but did not change the immunostaining properties of the PLP C-terminus. These results provide evidence that the carboxyl-terminus of PLP is located at the cytoplasmic face of oligodendroglial membranes.     

Glucocorticoid-induced upregulation of proteolipid protein and myelin-associated glycoprotein genes in C6 cells

The effect of dexamethasone on the expression of proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) genes was investigated in rat C6 glioma cells. The steady state level of the respective mRNA was quantitated by Northern blot analysis. The treatment of cells with dexamethasone transiently upregulated the expression of both genes with peak mRNA levels of approximately 10-fold over control levels occurring at day 3 for the PLP gene and at day 5 for the MAG gene. The effect was directly related to the drug concentration in the range from 10^?9 to 10^?5M. Combined exposure of the cells to dexamethasone and retinoic acid featured an additive effect on PLP gene expression, whereas MAG gene expression was depressed below detectability level. The dissimilarity in the response of the genes to dexamethasone and retinoic acid supports the contention that the genes are controlled by different mechanisms. Furthermore, the results indicate that the effects of dexamethasone and retinoic acid on the myelin genes are mediated by different regulatory pathways. © 1994 Wiley-Liss, Inc.     

Humoral immune recognition of proteolipid protein (PLP)-specific encephalitogenic epitopes in the SJL/J mouse

SJL/J mice were immunized with human PLP as well as encephalitogenic PLP peptides 139–151 and 178–191 and the resulting antibody responses examined for immunochemical specificity employing a panel of 17 synthetic PLP peptide ligands. All animals had demonstrable circulating titers of antibodies early in the humoral immune response to their respective encephalitogens, however, there was no clear qualitative correlation between antibody responses and the induction of EAE. In the majority of PLP immunized animals, determinant-specific antibody populations, including those against encephalitogenic centers, were not detectable in the presence of an anti-PLP antibody response. Antiencephalitogenic peptide antibodies were present in both 139–151 and 178–191 immunized animals regardless of clinical/histologic status. Neither group produced cross-reactive anti PLP antibodies as detected by ELISA. In animals immunized with peptide 139–151, only anti-139–151 antibody specificities were noted. In contrast, all animals immunized with peptide 178–191 had an antibody population cross-reactive with three other PLP peptides: 97–110, 209–217, and 215–228. As humoral immune responses can be demonstrated against PLP-specific encephalitogenic epitopes, the significance of these B cell responses should be considered in the context of their potential role in the development, modulation, and/or potentiation of EAE. © 1994 Wiley-Liss, Inc.     

Cross-reactivity between peptide mimics of the immunodominant myelin proteolipid protein epitope PLP139-151: comparison of peptide priming in CFA vs. viral delivery

Epidemiological evidence suggests that pathogens may trigger the development of autoimmune diseases such as multiple sclerosis (MS). Pathogens may trigger disease via molecular mimicry, wherein T cells generated against foreign epitopes are also cross-reactive with self-epitopes. Five pathogen-derived molecular mimics of PLP(139-151) (the immunodominant CD4(+) T cell myelin epitope in SJL mice) were previously identified. This study examines the degree of cross-reactivity between the different mimics, comparing mice primed with mimic peptide in CFA with mice infected with recombinant mimic-expressing viruses. The pattern of in vitro reactivity and ability to induce CNS disease differs between peptide priming and virus infection.     

Retinoic acid-regulated expression of proteolipid protein and myelin-associated glycoprotein genes in C6 glioma cells.

The effect of retinoic acid (RA) on the expression of myelin-specific genes, i.e., proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) in rat glioma C6 cells, was analyzed by Northern blot hybridization. RA-treatment increased the steady-state level of the PLP-specific messages within one day after RA administration and the upregulation reached a maximum on the third day. Concomitantly, the expression of MAG-specific messages in the RA-treated C6 cells dropped below the detectability limit. The expression of the PLP gene was directly related to the RA concentration increasing to approximately 44-fold over the control (untreated cells) level at 10(-6) M RA. The stimulatory effect was vitiated by cycloheximide indicating the involvement of intermediate genes in the PLP gene activation. The total cellular RNA content and the level of cyclophilin mRNA was not changed by the RA-treatment. The present data indicate that RA can be a potent modulator of the myelin-specific gene expression. Furthermore, the reciprocal response of PLP versus MAG genes to RA demonstrates that these two genes utilize different regulatory mechanisms.     

DM20 mRNA splice product of the myelin proteolipid protein gene is expressed in the murine heart.

Northern blot analysis of poly A(+) RNA isolated from mouse heart revealed the expression of 3.3 and 2.4 kb mRNAs that hybridized with a cDNA for the mouse proteolipid protein (PLP). In order to examine the relationship of these RNAs to the myelin PLP/DM20 mRNAs, a mouse heart cDNA library was prepared and screened with a mouse PLP cDNA. A cDNA was isolated, sequenced, and found to encode the DM20 variant of PLP. Polymerase chain reaction (PCR) analysis of heart cDNA with three sets of primers confirmed the presence of DM20 mRNA in mouse heart and indicated that it is the major splice product of the PLP gene expressed in that tissue. In situ hybridization localized the expression of the DM20 mRNA to the myocardial cells. Northern blot analysis indicated that expression of the DM20 mRNA is developmentally regulated in the murine heart, increasing significantly in concentration after 12 days postpartum. Northern analysis also revealed the expression of the DM20 mRNA in the hearts of the jimpy and quaking mutants. These results indicate that the PLP gene is expressed in tissues other than brain and support the concept that products of the PLP gene may have some biological role other than as structural components of myelin.     

Conservative amino acid substitution in the myelin proteolipid protein of jimpymsd mice.

The capacity for synthesizing and maintaining a compact myelin sheath is destroyed in a number of inborn errors of myelin metabolism. One class of hypomyelinating mutations, which displays an X-linked pattern of inheritance, is distinguished by marked disturbances in oligodendrocyte differentiation. We have defined the molecular defect in one such mutant that lacks mature oligodendrocytes, the X-linked jimpy myelin synthesis deficient (jpmsd) trait in mice. The structure of the gene encoding the most abundant myelin protein, proteolipid protein (PLP), was determined by mapping and partially sequencing genomic clones from jpmsd and wild-type mice. Jpmsd mice have a single base change in PLP, a C----T transition in exon 6 that would substitute a valine for alanine in both PLP and its alternatively spliced isoform, DM20. The mutation was confirmed by polymerase chain reaction-amplifying exon 6 from genomic DNA and then either sequencing the amplified DNA or directly probing exon 6 with oligonucleotides designed to detect a single base mismatch. The conservative amino acid replacement in PLP/DM20 of jpmsd mice results in a pleiotropic phenotype similar to that observed for the allelic mutation jimpy, in which a splicing defect has radically altered the PLP/DM20 protein. The accelerated turnover of oligodendrocytes in both mouse mutants suggests a function for PLP/DM20 in oligodendrocyte differentiation distinct from the role of these proteolipid proteins as structural components of the myelin sheath.     

Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis

T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for the rabbit.

A late onset, demyelinating form of experimental allergic encephalomyelitis (EAE) was induced in New Zealand White rabbits following immunisation with a synthetic peptide representing the amino acid sequence 91-110 of bovine myelin proteolipid protein (PLP). Histologically, disease was associated with varying degrees of central nervous system (CNS) inflammation, which in six of seven animals was accompanied by axonal degeneration and secondary demyelination. Primary demyelination with axonal sparing was generally absent in this model of EAE. Immunohistochemical and immunoelectron microscopic analysis of CNS tissue indicate that B cell epitopes within this encephalitogenic PLP sequence are not exposed at the surface of the myelin sheath, but are sequestered within compact multilamellar myelin. Furthermore, no correlation was observed between the anti-PLP antibody titer induced by the peptide and either the clinical severity or histopathology of the disease. These observations suggest that the B cell response to epitopes within the PLP sequence 91-110 does not play a primary role in the immunopathogenesis of PLP-induced EAE.