Proteolipid Protein Transcripts Research Articles

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP-mediated pathways. In this study, we investigated whether n-3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2-day-old rats, which were then killed after 3 days post-injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin-specific mRNAs was analyzed by real-time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA- and EPA-treated animals, but the effect was more pronounced in EPA-treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA-treated rats. A further indicator of accelerated myelination was the increase in 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) protein levels. In EPA-treated rats, the increased expression of myelin genes coincided with a decrease of cAMP-response element-binding protein (CREB)-DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA-treated rats was less pronounced than in controls. The down-regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n-3 PUFA beneficial effects on several demyelinating diseases.

Relevance of combinatorial profiles of intermediate filaments and transcription factors for glioma histogenesis

In order to define specific markers for histogenesis of three well-characterized subgroups of human gliomas (pilocytic astrocytomas, glioblastoma multiforme and oligodendrogliomas), we studied the expression of relevant markers that characterize gliomagenesis, by immunohistochemistry and in situ hybridization. They include the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin and nestin, the transcription factors Olig2, Nkx2.2 and Sox10, and the proteolipid protein transcripts plp/dm20. We show that the three major categories of human gliomas express a combinatorial profile of markers that gives new insights to their histogenesis and may help diagnosis. Pilocytic astrocytomas strongly express GFAP, vimentin, Olig2, Nkx2.2 and Sox10 but not nestin. In contrast, glioblastomas strongly express GFAP, vimentin and nestin but these tumours are heterogeneous regarding the expression of the transcription factors studied. Finally, in oligodendrogliomas, intermediate filament proteins are generally not observed whereas Olig2 was found in almost all tumour cells nuclei while only a subpopulation of tumour cells expressed Nkx2.2 and Sox10.

Cloning and expression of the proteolipid protein DM20 cDNA from the brain of the rainbow trout, Oncorhynchus mykiss

The myelin sheath in higher vertebrates consists predominantly of proteolipid protein (PLP) and its smaller isoform DM20. Mutations in the PLP gene produces several neurological disorders such as Pelizaeus-Merzbacher disease and the rumpshaker phenotype in mice. This paper describes the cloning and expression of DM20 from the brain of Rainbow trout. We have isolated a nearly full-length cDNA clone containing 1835 bp that codes for a protein of 258 amino acids. Trout DM20 shows extensive homology with DM20 from higher vertebrates and includes the four hydrophobic regions that are believed to span the myelin membrane. The DM20 transcript is expressed throughout the central nervous system of the trout but appears at its highest levels in the spinal cord and medulla oblongata. The transcript is expressed at very low levels on hatching day but increases 179-fold by the 5th week. Contrary to higher vertebrates, there is no switch to the PLP transcript in maturing trout. Moreover, the rsh mutation (186 Thr to Ile) that produces the rumpshaking neurological disorder in mice has no effect in trout.

Plasticity in the adult human oligodendrocyte lineage

Preoligodendrocytes have been described in cultures and tissue prints of adult human white matter (Armstrong et al., 1992). To characterize further these precursors of human oligodendrocytes, we have investigated whether they express genes playing a critical role in oligodendrocyte development. In the intact human brain, platelet-derived growth factor receptor alpha (PDGF alpha R) and myelin transcription factor 1 (MyTI) transcripts are expressed in 1-2% of cells of the oligodendrocyte lineage (OL), and clusters of such cells can be found in the periventricular region. Myelin basic protein transcripts containing exon 2 information (exon 2+ MBP), which are characteristic of the premyelinating stage, are detected in 15-20% of OL cells in vivo. When OL cells are separated from human white matter and allowed to regenerate in vitro, a much larger proportion of these cells express developmentally regulated genes, while exon 2- MBP and proteolipid protein (PLP) transcripts characteristic of mature OL cells appear transiently downregulated. Basic fibroblast growth factor (bFGF), even in the presence of PDGF, does not promote DNA synthesis in these cultured OL cells. Yet bFGF induces human oligodendrocytes to regenerate their processes rapidly in vitro and to express O4 antigens as well as exon 2+ MBP, MyTI, and PLP transcripts. While bFGF accelerates early regenerative processes, it also maintains high expression of exon 2+ MBP transcripts in OL cells for up to 2 weeks in vitro. In contrast, high levels of insulin in the absence of bFGF allow accumulation of exon 2- MBP and PLP transcripts in most OL cells at 2-3 weeks in vitro. We propose that the myelinated human brain harbors a small pool of precursors of oligodendrocytes and that growth factor-regulated phenotypic plasticity rather than mitogenic potential accounts for the regeneration of oligodendrocytes in the initial stages of demyelinating diseases such as multiple sclerosis.

Transcriptional regulation of the rat PLP promoter in primary cultures of oligodendrocytes.

Proteolipid protein (PLP) is the major intrinsic membrane protein of CNS myelin and is expressed in oligodendrocytes as part of a coordinate program of myelin-specific gene activation. In order to identify the DNA sequences and proteins involved in the regulation of PLP transcription, we have analyzed the 5' flanking sequences of the rat PLP gene by transient transfections into primary cultures of developing oligodendrocytes, the glial tumor line, C6, and L cells. High levels of expression of the CAT reporter gene in oligodendrocytes and C6 cells were obtained with constructs containing both 4270 and 225 bp of PLP promoter. A fusion construct containing 1061 bp of the PLP promoter, however, showed two-fold lower CAT expression. In addition, the activity of these promoter fusion constructs in oligodendrocytes was 2.5-4.6 higher than that observed in C6 cells, while very little expression was found in L cells. These data suggest that 225 bp of PLP promoter is sufficient for oligodendrocyte-specific regulation of PLP expression. Furthermore, both positive and negative elements within the PLP promoter are involved in this process. Finally, primary cultures of developing oligodendrocytes are a useful model system for the analysis of myelin-specific gene activation.

Appearance of PLP mRNA in Specific Regions of the Developing Rat Lumbosacral Spinal Cord as Revealed by in Situ Hybridization

The capacity of specific regions and fiber tracts of developing rat lumbosacral spinal cord to express proteolipid protein (PLP) mRNA was examined using in situ hybridization. PLP mRNA was undetectable at Day 18 postfertilization but present in the white matter by Postnatal Day 0 (P0). Initially, levels of PLP mRNA were higher in ventral than in dorsal funiculi. Hybridization signal was observed in the corticospinal tract by P10. Appearance of PLP message in gray matter lagged several days behind that in white matter. The expression of PLP mRNA in gray matter rose between P10 and P40 and the abundance of PLP transcript remained high in white matter during the same period. In the adult spinal cord some oligodendrocytes in the Lissauer tract and in the posterior commissure expressed low levels of PLP mRNA, which was undetectable in the substantia gelatinosa. Comparing the time of emergence of PLP mRNA to the histological appearance of myelination gave evidence that the message is expressed in oligodendrocytes that are not many days away from myelin formation. These results provide new information on the topographic distribution of PLP mRNA during myelination of the rat spinal cord.

Axons modulate the expression of proteolipid protein in the CNS.

We examined the expression of mRNA encoding proteolipid protein (PLP), the major myelin protein in the CNS, in developing rat cerebrum, and in normal and degenerating optic nerves. PLP transcripts were initiated at two clusters of start sites that were separated by about 30 base pairs. During the peak of PLP mRNA expression in developing cerebrum, a higher proportion of PLP transcripts were initiated from the distal start site, furthest from the open reading frame, than in mature cerebrum. We enucleated one eye of immature rats to cause Wallerian degeneration in the optic nerve. In these degenerating optic nerves, the steady state levels of PLP mRNA fell markedly, and the proportion of distally initiated PLP transcripts declined to the same proportion found in normal adult nerves. Changes in myelin gene expression were not limited to PLP mRNA, as the steady-state levels of myelin basic protein (MBP) mRNA paralleled those of PLP mRNA in the developing cerebrum and in degenerating optic nerves. Thus, oligodendrocytes require axons to maintain their normal levels of PLP and MBP transcripts and the high proportion of distally initiated PLP transcripts that characterize early myelination.

DM‐20 mRNA Is Expressed During the Embryonic Development of the Nervous System of the Mouse

We used both the polymerase chain reaction (PCR) and in situ hybridization to search for the presence of proteolipid protein (PLP) gene transcripts in the developing mouse. Total brain RNA extracted from 13-19-day embryos, analyzed by PCR, demonstrated the presence of a single transcript that was unambiguously identified with the DM-20 mRNA. RNA samples from postnatal day 2 animals also showed a signal corresponding to the PLP transcript, in addition to the DM-20 message. By in situ hybridization of 10-day embryos using a DM-20 antisense cRNA probe, we showed that the localization of the DM-20 message was restricted to the diencephalic basal plate. On the same embryo sections, in addition to the brain localization, an intense hybridizing signal was also detected in the trigeminal and spinal ganglia, the vagal glossopharyngeal ganglion, and the sympathetic ganglion chain. The demonstration of transcription of the PLP gene, long before the beginning of the myelination process, suggests that in addition to a structural function in myelin compaction, some of the products of the PLP gene (DM-20) may have a role during the compartmentalization and differentiation of the neural tube.

Structure and expression of proteolipid protein in the peripheral nervous system.

Proteolipid protein (PLP), the major myelin protein in the central nervous system (CNS), is also made by Schwann cells (SC) in the peripheral nervous system (PNS) but is not incorporated into the SC myelin sheath. We analyzed several PLP cDNA clones isolated from a rat sciatic nerve cDNA library and found that their coding sequences were identical to PLP cDNAs previously isolated from the CNS. In addition, we have discovered an unusual form of PLP message, present in both brain and sciatic nerve RNA, that is likely formed by alternative splicing within the 3' untranslated region of the primary PLP transcript. The absence of PLP from the SC myelin sheath thus cannot be explained by an alteration in its amino acid sequence. Steady-state levels of PLP mRNA in SC cultures treated with the cAMP analogue dibutyryl cAMP (dBcAMP) were not increased, whereas dBcAMP increased steady-state levels of mRNA encoding the major myelin protein, P0. We have also shown that expression of PLP, unlike that of P0, is regulated in SC in vitro at a posttranscriptional level. Finally, the steady-state levels of P0 mRNA are much more dramatically reduced than those of PLP mRNA during Wallerian degeneration of the peripheral nerve. Thus PLP expression in the PNS is probably controlled by different molecular mechanisms from P0, and may not be part of the coordinate program of myelin gene expression. In contrast to its expression in the PNS, transcription of PLP in the CNS is coordinately regulated along with the other myelin protein genes, suggesting there may be differences in the cis-acting elements and transacting factors involved in the regulation of PLP transcription in SC and oligodendrocytes (OC). Consistent with this notion, we have found that most PLP transcripts are initiated at the more proximal of two start sites in the PNS, while in the CNS proportionally more PLP transcripts are initiated from the distal start site. We propose that the proximal site, utilized predominantly in SC, is responsible for maintenance expression of PLP and is not inducible, while the distal site is responsible for the rapid, inducible increase of PLP message during brain development.

Levels of Proteolipid Protein mRNAs in Peripheral Nerve Are Not Under Stringent Axonal Control

The proteolipid protein (PLP) is the major protein in the myelin sheath of the CNS. It was recently reported that PLP coding transcripts are also found in the PNS, although the protein was not detectable in peripheral nerve myelin. In the present investigation, levels of mRNA for PLP in sciatic nerve were studied during development and following transection and crush injury. Results were compared to those for P0, the major PNS myelin protein, and the myelin-associated glycoprotein (MAG). PLP transcript levels were very low at 21 days in sciatic nerve and remained unchanged in the adult sciatic nerve. This contrasts markedly with P0 and MAG mRNAs, which are expressed at high levels during development and decrease in content significantly by adulthood. The level of PLP messages was reduced approximately 40% in the quiescent Schwann cells in the distal segment of the sciatic nerve at 21 days after permanent transection, yet P0 mRNA levels were very low, and MAG mRNAs were undetectable in this tissue. The distal segment of the crush-injured sciatic nerve is characterized by transient demyelination followed by rapid myelination. PLP mRNA levels remained comparatively unaffected in the 3-week period following crush injury. RNase protection experiments using two antisense riboprobes confirmed that levels of PLP-derived protected fragments, corresponding to PLP and DM-20 messages, remained unchanged in the developing and adult sciatic nerve. These results indicate that myelin-specific P0 and MAG genes are tightly controlled at the level of transcription through Schwann cell-axonal interactions, whereas PLP transcription in the peripheral nerve remains nearly dissociated from axonal influences.

Developmental Expression of Myelin Protein Genes in Dysmyelinating Mutant Mice: Analysis by Nuclear Run‐Off Transcription Assay, In Situ Hybridization, and Immunohistochemistry

Gene expression for myelin proteolipid protein (PLP) and myelin basic protein (MBP) in the dysmyelinating mutant mice shiverer and jimpy was analyzed by nuclear run-off transcription assay, in situ hybridization, and immunohistochemistry. The level of PLP transcription in shiverer brains was lower than that in controls at postnatal day 18 but relatively higher at later stages. In spite of the considerable amount of hybridization with PLP cDNA, immunoreaction for PLP was greatly reduced in shiverer mice throughout their lives, probably owing to a defect in the assembly of PLP into myelin. Abnormal deposition of PLP in oligodendroglial cell bodies suggested that transport of PLP to myelin is delayed in shiverer brains. The number of oligodendrocytes expressing PLP mRNA was drastically reduced in jimpy mice. MBP mRNA in jimpy mice is localized preferentially in oligodendroglial cell bodies, a result suggesting that oligodendrocytes in jimpy are mostly the immature type. Although transcriptional activity of the MBP gene in jimpy was greatly reduced, a finding reflecting the decrease in the number of mature oligodendrocytes, that of the PLP gene remained high at early stages. The discrepancy of the two gene expressions is discussed relative to the role of PLP transcripts at early stages of myelination.

Myelin-deficient rat: a point mutation in exon III (A----C, Thr75----Pro) of the myelin proteolipid protein causes dysmyelination and oligodendrocyte death.

The expression of the proteolipid protein (PLP) gene of the myelin deficient (md) and normal rat was studied during the myelination period. The sizes of the PLP transcripts (1.6 and 3.2 kb) in the md and normal rat were identical although the md PLP messenger RNA level was extremely reduced as shown by in situ hybridization and Northern blot hybridization analysis. The structure of the md proteolipid protein gene was analyzed on the cDNA and genomic level. The molecular basis of the myelin deficiency phenotype has been elucidated: a point mutation in exon III (A----C transversion) verified by cDNA and genomic DNA sequencing causes a mutation of Thr75 to Pro and creates an additional AvaII restriction site in exon III of the md rat. The threonine to proline mutation located within the second transmembranal alpha-helix might induce a conformational change and thereby prohibit the integration of PLP into the membrane with the clinical manifestation of dysmyelination leading to premature death within 3-6 weeks.

In situ hybridization analysis of myelin gene transcripts in developing mouse spinal cord

We analyzed the location and abundance of transcripts for the 4 CNS myelin protein genes, myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide phosphohydrolase (CNP), in the mouse cervical spinal cord from the time of rapid myelination until adulthood (8-45 d). In the white matter, maximal levels of transcripts were found for each of the myelin genes at the peak of myelination (8 d). Total MBP and PLP mRNAs stayed high until 20 d and showed a minor decrease thereafter. In contrast, MAG and the MBP exon 2 containing transcripts (coding for the 21.5 and 17 kDa MBP isoforms) decreased sharply between 8 and 20 d, suggesting that high levels of these transcripts are needed primarily during the initiation of myelination. CNP transcripts were less abundant, maintained high expression until 20 d, and then decreased sharply. PLP, MAG, and CNP transcripts were clustered in the oligodendrocyte cell body, while MBP mRNAs were scattered throughout the cell body and processes. In contrast to the white matter, all these myelin specific transcripts in the gray matter showed a marked increase from 8 to 20 d, as did the number of oligodendrocytes identified by CNP immunostaining. MAG transcripts were found in white matter and in satellite and other oligodendrocytes of the gray matter but not in neurons identified by their expression of neurofilament transcripts. The results of our quantitative in situ hybridization study are in good agreement with those of previous molecular studies and provide new information on the cellular and topographic distribution of myelin-specific mRNAs during myelination.

Developmental expression of neural cell type‐specific mRNA markers in the myelin‐deficient mutant rat brain: Inhibition of oligodendrocyte differentiation

We have studied gene expression of neuroglial cell markers in the myelin-deficient (md) rat brain during postnatal development. Northern blots and slot blots of poly(A)+ RNA from developing brain were sequentially probed with cDNAs specific for the oligodendrocyte markers glycerol phosphate dehydrogenase (GPDH), myelin basic protein (MBP), and proteolipid protein (PLP), for the neuronal marker glutamic acid decarboxylase (GAD), and for the astrocyte markers glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). GPDH mRNA levels were also examined in two peripheral tissues, liver, and skeletal muscle (hindlimb). Despite a lack of CNS myelin in the md mutant, transcripts of all oligodendroglial markers were detectable except the 1.6-kb PLP message. Brain GPDH mRNA levels were initially equivalent in md and unaffected littermates at postnatal day 15 (PI5), but the mutants failed to display the normal developmental increase in gene expression. By P25, GPDH mRNA expression in md rat brain was approximately 20% of control levels. GPDH mRNA expression in peripheral tissues was less affected than in brain and was lower in md mutants only at the later developmental ages. Expressions of GAD, GFAP, and GS mRNAs in developing md rat brain were not altered. The mRNA levels of the two myelin markers, MBP and PLP, were severely impaired in md rat brain during the entire myelinating period and represented less than 10% of control mRNA levels at P25. The most important observation was that the large PLP transcript (3.2 kb) was slightly shorter in size in md rat brain as compared to normals.(ABSTRACT TRUNCATED AT 250 WORDS)

Splice site selection in the proteolipid protein (PLP) gene transcript and primary structure of the DM-20 protein of central nervous system myelin.

Proteolipid protein (PLP) is the major myelin membrane protein of the central nervous system. We have isolated a copy of an alternatively spliced PLP gene transcript from a mouse brain cDNA library that was screened for PLP-related sequences. The encoded 241-amino acid protein differs from PLP by an internal deletion of 35-amino acid residues (116-150) from the major hydrophilic domain. This PLP variant is identical with the DM-20 protein of myelin, previously described as a brain-specific myelin component and known to be related to PLP. We determined the corresponding nucleotide sequence of the rat PLP gene and found that DM-20 mRNA results when a second 5' splice site, located 105 nucleotides within the third exon of the primary PLP transcript, is utilized in precursor mRNA (pre-mRNA) splicing. This demonstrates that alternative 5' splice site selection can determine the protein product of a cellular gene. DM-20 mRNA is expressed in rat brain with approximately 50% abundance relative to PLP mRNA and appears to be developmentally coregulated.

Human myelin DM-20 proteolipid protein deletion defined by cDNA sequence

Using a myelin proteolipid protein (PLP) cDNA as probe, a 1kb cDNA was isolated from a human retinal cDNA library. This clone, designated pDM-20, contained all of the coding sequence of PLP except for 105 base pairs which encode amino acids 117-151 of PLP. This deletion would result in a protein of the size of DM-20, the second major proteolipid of Central Nervous System (CNS) myelin. Homology between the DNA sequence at the 5' end of the deletion and the donor splicing consensus sequence suggests that PLP and DM-20 transcripts are derived from a single PLP gene by alternate splicing.

Aberrant splicing of proteolipid protein mRNA in the dysmyelinating jimpy mutant mouse.

cDNA clones encoding proteolipid protein (PLP) were isolated from a mouse brain library and sequenced. We describe two transcripts arising from the PLP locus by alternative splicing: the major one encodes the 277-amino acid PLP protein and the minor one corresponds to the DM-20 protein, a PLP-like protein of 20,000 Mr that shares both amino and carboxyl regions with PLP. These two transcripts lack approximately 70 bases in PLP mRNA from the dysmyelinating jimpy mutant. The deletion spans amino acids 208-232; however, this region is present in the jimpy PLP-encoding gene. We propose that the jimpy mutant suffers a point mutation or the deletion of a few bases in the PLP gene that alters the normal splicing pattern and generates partially deleted PLP transcripts.