Mouse Myelin Basic Protein Research Articles

A novel transcript overlapping the myelin basic protein gene.

Myelin basic protein (MBP) is a major constituent of myelin synthesized by oligodendrocytes and Schwann cells. We have investigated the expression of mouse MBP RNAs outside the nervous system. Nuclease protection experiments indicate that RNAs containing exon 1 and not the six downstream exons of the MBP gene are transcribed in various hemopoietic tissues. We have isolated a hemopoietic MBP-related (HMBPR) cDNA clone from a mouse bone marrow cDNA library screened with an MBP cDNA probe. This clone contains exons 1a and 1b and a part of intron 1 of the MBP gene. An additional 5' region, encoded by at least three unidentified exons, lies upstream of exon 1a. The HMBPR clone corresponds to a 5-kb RNA expressed in bone marrow, spleen, thymus, and macrophagic cells. This transcript is expressed at a similar level in brain, although at a lower level than the classical 2-kb mRNA. These data indicate that a new transcript, overlapping the MBP transcription unit and controlled by a distinct promoter, is expressed in hemopoietic tissues. This RNA might encode a 21-kDa protein sharing a common domain with MBP.

Amelioration of autoimmune encephalomyelitis by myelin basic protein synthetic peptide-induced anergy.

Experimental autoimmune encephalomyelitis (EAE), a demyelinating disease of the central nervous system that can be induced in susceptible strains of mice by immunization with myelin basic protein (MBP) or its immunodominant T cell determinants, serves as a model of human multiple sclerosis. Tolerance to MBP in adult mice was induced by intraperitoneal injection of synthetic peptides of immunodominant determinants of MBP and prevented MBP-induced EAE. Furthermore, tolerance-inducing regimens of peptides administered to mice after the disease had begun (10 days after induction with MBP) blocked the progression and decreased the severity of EAE. Peptide-induced tolerance resulted from the induction of anergy in proliferative, antigen-specific T cells.

Subjugation of dominant immunogenic determinants within a chimeric peptide.

The mechanism of immunodominance was investigated using chimeric peptides from mouse myelin basic protein consisting of the immunodominant I-Au-restricted Ac1-11, attached by a peptide bond to I-Eu-restricted 35-47. Our results indicate that this chimeric peptide and certain of its derivatives were excellent immunogens both in vitro and in vivo. Notably, on immunization with Ac1-11:35-47 or Ac1-11 (Ala4):35-47, the proliferative T cell responses to each of its component peptides were almost completely "subjugated" in favor of neo-determinants that are I-Eu restricted. Furthermore, each of 11 hybridomas derived after immunization with Ac1-11:35-47 had specificity for junctional neo-determinants and none could be stimulated to produce interleukin-2 from Ac1-11 or 35-47. Subjugation of the immunogenicity of the original determinants occurred regardless of their dominance when separate. It did not appear to result from non-availability of the original determinants because the chimeric peptide was able to induce neonatal tolerance to each of its constituents. These results indicate that in an overlapping multideterminant array, the dominant determinant is unpredictable from historical data about any of the components. Determinant choice, at any stage of processing, may be governed by competitive aspects of determinant capture in an environment where all components--antigen, major histocompatibility complex and T cell receptor--are available.

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. V. Hierarchy of suppression by myelin basic protein from different species

We have been investigating the suppression of experimental autoimmune encephalomyelitis (EAE) by oral tolerization to autoantigens. In the present study the tolerizing effect of orally administered myelin basic protein (MBP) from different species was examined in the Lewis rat, Hartley guinea pig, and SJL/J mouse model of EAE. Animals were fed guinea pig, rat, bovine, human or mouse-BMP and them immunized with the homologous species of MBP or myelin: Lewis rats were immunized with rat MBP, Hartley guinea pigs with guinea pig-MBP, and SJL/J mice with mouse myelin. Clinical expression of EAE and delayed-type hypersensitivity (DTH) responses to MBP were assessed. In each species, suppression of disease and DTH responses were most pronounced by tolerization with the homologous species of MBP. In addition, cross-species tolerization was observed in each species and in general was less suppressive than homologous MBP although in some instances MBP from a heterologous species was as effective as tolerization with the homologous species. We also studied guinea pig-MBP induced EAE in the Lewis rat because it is widely studied model of EAE and found that oral tolerization with guinea pig MBP was as suppressive as rat MBP. Of note is that oral tolerization with mouse MBP suppressed myelin-induced EAE in the SJL mouse in which autoimmunity to proteolipid protein appears to play a primary role, suggesting that antigen-driven bystander suppression following oral tolerization with autoantigens (Miller et al., 1991b) may be an important contributing mechanism for suppression of EAE following oral tolerization with MBP in this model. Taken together, these results suggest that epitopes of MBP distinct from the encephalitogenic region participate in triggering suppression following oral tolerization and have been important implications for treating human diseases by oral tolerization to autoantigens.

Cell and tissue-specific expression of a heterologous gene under control of the myelin basic protein gene promoter in transgenic mice

Myelin basic protein (MBP) is the second most abundant protein in CNS myelin. We have used transgenic mice to investigate the ability of the 5′ flanking sequence of the mouse MBP gene to regulate the cell-type-specific- and temporal expression of a heterologous gene under its control. Transgenic mice were produced with a construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene downstream of the MBP 5′ flanking sequence and CAT expression was monitored both enzymatically and histochemically. The results indicate that 1323 bp of 5′ flanking is sufficient to direct CAT expression specifically to the tissue and cell-type, in which MBP is normally synthesized. Additionally, this length of the sequence also retains the ability to temporally regulate CAT levels in a manner analogous to endogenous MBP levels.

Molecular basis for oligodendrocyte degeneration in jimpy mouse

The mouse proteolipid protein (PLP) gene was cloned into the λ bacteriophage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the PLP gene started from multiple sites. There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region.Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression. Homology search against the mouse MBP gene revealed that several boxes in the 5′-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5′-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system.PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5′-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level.

Effects of chronic administration of antidepressants on 3H-flunitrazepam binding in rat brain

The mouse proteolipid protein (PLP) gene was cloned into the λ bacteriophage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the PLP gene started from multiple sites. There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region.Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression. Homology search against the mouse MBP gene revealed that several boxes in the 5′-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5′-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system.PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5′-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level.

Sequences involved in brain-specific in vitro transcription from the core promoter of the mouse myelin basic protein gene

In the previous study, we have shown that a short DNA stretch from -35 to -17 in the core promoter of the mouse myelin basic protein (MBP) gene is mainly responsible for brain-specificity in in vitro transcription. In this study, we found from intensive mutation analysis that the TATA-box sequence at -34 is not critical, but sequences downstream from the TATA-box especially around -20 were important for brain-specificity. The existence of a tissue-specific factor for the MBP core promoter different from the TATA-box-binding factor TFIID is implicated.

Involvement of the Nuclear Factor I Motif in the Mouse Myelin Basic Protein Promoter in Cell-Specific Regulation: Comparison with the Mouse Glial Fibrillary Acidic Protein Promoter: (Myelin basic protein/Glial fibrillary acidic protein/Nuclear factor I/Transcription/Cell specificity).

We investigated the cis-element for cell-specific expression in the mouse myelin basic protein (MBP) promoter by transfection assay in comparison with that of the glial fibrillary acidic protein (GFAP) promoter. The MBP promoter region including the 1.3 kb upstream region from the transcription start point was found to be highly expressed in NG108-15 cells, a nervous tissue-derived hybrid cell line, but not in fibroblasts. In contrast, the GFAP promoter region including the region 2.6 kb upstream of the transcription start point was found to function dominantly in C6 glioma cells, but not in NG108-15 cells. Deletion analysis revealed that the NFl motif in the MBP promoter (named MBTE) resulted in the loss of preferential expression in NG108-15 cells. The GFAP promoter also possessed another NFI motif (named GFII) and its distance from the transcription start point was very similar to that of the MBP promoter. Base-substitution mutation of GFII to MBTE in the GFAP promoter caused remarkable increase in GFAP promoter activity in NG108-15 cells but only slight increase in it activity in C6 cells. These results suggest that the NFI motif in the MBP promoter (MBTE) functions as a cis-acting promoter element that governs cell-specific transcription.

Identification of the New Isoforms of Mouse Myelin Basic Protein: The Existence of Exon 5a

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.

Demonstration of a transcription element in vitro between the capping site and translation initiation site of the mouse myelin basic protein gene

A transcription element was identified, by in vitro analyses, just downstream from the capping site of the mouse myelin basic protein (MBP) gene. Deletion of this element caused a dramatic drop of transcription efficiency in mouse brain, rat liver and HeLa cell nuclear extracts, regardless of the form of DNA being closed circular or linear form. DNase I footprint analysis demonstrated the presence of a ubiquitous trans-acting factor for this region. This element functioned even when it is located in the normal direction downstream from the adenovirus major late promoter. Mutation analysis suggested that an essential part of the downstream element was located between +25 and +45.

Acute Experimental Allergic Encephalomyelitis in SJL/J Mice Induced by a Synthetic Peptide of Myelin Proteolipid Proteina

Clinical, histologic, and ultrastructural characteristics of acute experimental allergic encephalomyelitis (EAE) induced by sensitization with a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 139-151 HCLGKWLGHPDKF were studied in SJL/J mice. Groups of mice were immunized with 20, 50, or 100 nmol of the peptide and were killed from seven to 28 days after sensitization or when they were moribund. Beginning on Day 9, the mice showed signs of EAE and the disease progressed rapidly to paralysis. Central nervous system (CNS) inflammation, edema, gliosis, and demyelination were found in all mice killed between Days 10 and 28 and white matter lesion areas correlated with clinical score at the time the mice were killed. Peripheral nerve roots and the cauda equina did not have lesions. Within the range studied, the severity of clinical or histologic disease was the same regardless of the PLP peptide dose. Two of ten mice immunized with 100 nmol and none of 14 mice given smaller doses of a synthetic peptide of mouse myelin basic protein (MBP) showed clinical EAE. These mice had small numbers of CNS lesions that were indistinguishable from those in PLP peptide-sensitized mice. These findings demonstrate that immunization of SJL/J mice with PLP peptide 139-151 produces a disease with the clinical and morphologic features of CNS tissue-, whole PLP-, whole MBP-, and MBP peptide-induced acute EAE. Thus, PLP is a major encephalitogen and immune reactions to epitopes of different myelin proteins may induce identical patterns of injury in the CNS.

Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

Expression of myelin basic protein (MBP) in mice is regulated in a cell- and stage-specific manner during brain development. The MBP control region contains multiple cis-acting elements, shown by in vivo and in vitro assays, which are responsible for its unique pattern of transcription. Using synthetic DNA fragments spanning the MBP control region, we have analyzed nuclear proteins obtained from newborn (2-3 d), young adult (18-30 d), and adult (60 d) animals; these nuclear proteins form DNA-protein complexes with the MBP regulatory region. Brain extracts from young adult and adult mice showed enhanced binding activities with the sequences supporting transcriptional activation in glial cells. Deletion analysis of the proximal activating sequence located at position -14 to -50 with respect to the RNA initiation site resulted in identification of a small region, located between nucleotides -14 to -37, which is required for formation of the complexes. Southwestern assay revealed a major 39-kD protein from young adult brain extract that recognizes the sequences between nucleotides -14 to -37. An additional minor 37-kD protein, derived from young adult brain extract, was also found to be associated with this proximal activating region. Of particular interest is the observation that the minor 37-kD protein became more abundant in the extract derived from adult brain, whereas the major 39-kD protein became less abundant. The possible role of these proteins in cell/stage-specific transcription of MBP is discussed.

Isolation of complementary DNAs encoding a cerebellum-enriched nuclear factor I family that activates transcription from the mouse myelin basic protein promoter.

A nuclear factor I (NFI) binding sequence in the myelin basic protein (MBP) promoter is required for brain-specific transcription. In the present study, we isolated multiple cDNAs encoding a family of NFI, NFI-B, from mouse brain cDNA libraries. These clones contain unique nucleotide sequences at their 5', 3', or middle portions, presumably as a result of alternative splicing. The entire sequence of NFI-B suggests that NFI-B is a mouse counterpart of NFI-L, which was previously isolated from rat liver. NFI-B is highly enriched in the cerebellum, in contrast with a ubiquitous NFI, CTF/NF-I. NFI-B proteins translated in vitro could bind to the MBP NFI site. Furthermore, we demonstrated by transfection experiments that NFI-B activated transcription from transfected MBP promoter in neuronal/glial NG108-15 cells.

Core promoter of the mouse myelin basic protein gene governs brain-specific transcription in vitro.

The core promoter of the mouse myelin basic protein (MBP) gene from -36 to +12 was preferentially transcribed in brain nuclear extracts. Both the TATA at -34 and downstream elements to +12 were required for efficient, accurate and brain-specific transcription. From brain and liver nuclear extracts, we have partially purified the general transcription factor TFIID. The partially purified fractions contained TATA element binding factors of the MBP promoter as well as adenovirus major late promoter (MLP). The tissue-derived TFIID was functionally exchangeable for the HeLa TFIID, and directed transcription from the MLP. Surprisingly, the brain TFIID activated transcription from the MBP core promoter while the liver TFIID did to a much lesser extent. Exchange of the TATA-containing short DNA stretch to the MBP core promoter for a corresponding region of the mouse albumin promoter or MLP abolished the brain specificity. We found that several tissue-specific promoters other than MBP, such as mouse neurofilament and human alpha-1-antitrypsin promoters were also transcribed much more efficiently by the brain and liver TFIID, respectively. We suggest that different tissues contain functionally non-equivalent TFIID or TFIID-like activities.

Conservation Throughout Vertebrate Evolution of the Predicted β‐Strands in Myelin Basic Protein

To identify functionally important parts of the 18.5-kDa myelin basic protein (MBP), the amino acid sequences from 10 species ranging from shark to human were aligned using the SEQHP computer program. The residues that are invariant or very conservatively substituted (Arg/Lys, Ser/Thr, Ile/Leu, Asp/Glu) among all 10 proteins were scored. Of the 72 conserved residues in the 170-residue human protein (42% conserved), 32 are found within the five beta-strands previously predicted (45 residues, 71% conserved), 23 within the small-loops region (42 residues, 55% conserved), but only 17 within the large-loops region (83 residues, 20% conserved). Of the 22 hydrophobic residues within the predicted beta-sheet of human MBP, 20 hydrophobic residues remain in the shark protein, 19 of them in the same positions. In contrast, there are 10 hydrophobic residues elsewhere in the human protein, but only 7 remain in the shark protein and only 1 of them is in the same position. The triprolyl sequence found in all mammalian MBPs and in the chicken MBP is not conserved in the shark protein. The four alternately spliced forms of mouse MBP can be accommodated by the beta-structural model, but not the 17-kDa human MBP, which lacks exon 5. These findings confirm the crucial role of the hydrophobic residues in the predicted beta-sheet for the structure and function of the protein. It seems likely that the conserved portions of the protein make an important contribution to the highly ordered lamellar structure of myelin.

Acute Experimental Allergic Encephalomyelitis in SJL/J Mice Induced by a Synthetic Peptide of Myelin Proteolipid Protein

Clinical, histologic, and ultrastructural characteristics of acute experimental allergic encephalomyelitis (EAE) induced by sensitization with a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 139-151 HCLGKWLGHPDKF were studied in SJL/J mice. Groups of mice were immunized with 20, 50, or 100 nmol of the peptide and were killed from seven to 28 days after sensitization or when they were moribund. Beginning on Day 9, the mice showed signs of EAE and the disease progressed rapidly to paralysis. Central nervous system (CNS) inflammation, edema, gliosis, and demyelination were found in all mice killed between Days 10 and 28 and white matter lesion areas correlated with clinical score at the time the mice were killed. Peripheral nerve roots and the cauda equina did not have lesions. Within the range studied, the severity of clinical or histologic disease was the same regardless of the PLP peptide dose. Two of ten mice immunized with 100 nmol and none of 14 mice given smaller doses of a synthetic peptide of mouse myelin basic protein (MBP) showed clinical EAE. These mice had small numbers of CNS lesions that were indistinguishable from those in PLP peptide-sensitized mice. These findings demonstrate that immunization of SJL/J mice with PLP peptide 139-151 produces a disease with the clinical and morphologic features of CNS tissue-, whole PLP-, whole MBP-, and MBP peptide-induced acute EAE. Thus, PLP is a major encephalitogen and immune reactions to epitopes of different myelin proteins may induce identical patterns of injury in the CNS.

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of 14C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117-157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a Kd similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.

Expression of a Novel Transcript of the Myelin Basic Protein Gene

A cDNA (M41) corresponding to a mouse myelin basic protein (MBP) mRNA with a longer 5'-untranslated region than predicted from earlier studies of MBP gene structure has been isolated and characterized. The additional 5'-untranslated region is encoded by two previously unidentified exons upstream of the major transcription start site of the gene. Using a DNA probe specific for M41-MBP mRNAs, Northern blot analysis indicated that expression of this transcript follows a developmental course in mouse brain similar to that of the majority of MBP mRNAs, but that the level of expression varies between brain and spinal cored. Expression of MBP mRNAs similar to the mouse M41-MBP also was identified in rat brain. The results suggest that the structure of the MBP gene is more complex than originally thought, containing at least two more exons. There appears to be at least one more MBP gene promoter that directs the synthesis of a subset of MBP mRNAs with a unique 5'-untranslated region.

Rapid preimplantation detection of mutant (shiverer) and normal alleles of the mouse myelin basic protein gene allowing selective implantation and birth of live young.

As a model for the detection of human genetic disease in preimplantation embryos, we describe a method in which trophectoderm biopsy samples from viable mouse blastocysts are simultaneously analyzed for the presence of a normal or mutant allele of the myelin basic protein gene by the polymerase chain reaction. The biopsied embryos are kept in culture during analysis of biopsied material and later reintroduced to a foster mother. Prenatal diagnosis can be completed in less than 7 hr. The identity of either amplification product was proved conclusively by direct sequence analysis of amplified products. Ninety-six percent of recovered blastocysts survived biopsy, as judged by re-formation of a blastocyst cavity in culture. Fifty-nine percent of the biopsied embryos established pregnancy by day 6.5, compared to 88% of unmanipulated controls. This approach can be applied to preimplantation diagnosis of human genetic diseases by using extraembryonic cells from blastocysts obtained after in vitro fertilization or uterine lavage. It will make possible the elimination of a mutant allele from a family in a single generation.